Supplementary Figures 1 through 8 from Molecular Radiotherapy Using Cleavable Radioimmunoconjugates That Target EGFR and γH2AX (2023)

&lt;p&gt;PDF - 1380KB, Supplementary Figure S1. Schematic overview of the synthesis of (A) non-cleavable anti-gH2AX-PNE and (B) cleavable anti-gH2AX-N-SS-E. Supplementary Figure S2. (A) MDA-MB-468 cultures were exposed to 123I-EGF for 2 h plus increasing concentrations of EGF, anti-gH2AX-PNE or anti-gH2AX-N-SS-E. (B) Extracts from irradiated, gH2AX-containing 231-H2N cells were coated onto a RIA plate and exposed to 123I-anti-gH2AX for 2 h plus increasing amounts of anti-gH2AX, anti-gH2AX-PNE or anti-gH2AX-N-SS-E. For both (A) and (B) the relative amount of 123I that bound to plates was determined and LogIC50 values were calculated. Results are shown as the mean of three replicates plus or minus SD (C) PAGE analysis of 1. unmodified anti-gH2AX, 2. anti-gH2AX-PNE and 3. anti-gH2AX-N-SS-E. Supplementary Figure S3. (A) Anti-gH2AX-N-SS-(123I-EGF) was exposed to 0 or 1 mM glutathione. Size exclusion chromatography was performed using a G50 sephadex minicolumn. The amount of 123I in each fraction was measured. (B) 123I-EGF was synthesized as described in the methods section. G25 size exclusion chromatography was then performed on 123I-EGF and the cleaved product resulting from (A). (C) To demonstrate that EGF is released from rIgG-N-SS-E, rIgG-N-SS-E was exposed to glutathione and purified by G50 size exclusion chromatography. Then, EGFR positive MDA-MB-468 cells were exposed to 123I-EGF for 2 h at 4{degree sign}C with or without addition of the low molecular weight peak product. Blocking of 123I-EGF binding to MDA-MB-468 cells show that at least some of the low MW fraction binds to EGFR. Supplementary Figure S4: (A) 231-H2N cells were exposed for 4 h to AF555-labeled anti-γH2AX-PNE (red), fixed, and mounted with DAPI (blue). (B) 231-H2N cells were exposed for 4 h to AF555-labeled anti-γH2AX-N-SS-E (red), fixed, and mounted with DAPI (blue). Supplementary Figure S5: SQ20b cells were exposed for 1 h to AF555-labeled anti-gH2AX-N-SS-E or rIgG-N-SS-E, and irradiated (4 Gy) or sham irradiated. After 4 or 24 h, cells were fixed, stained for gH2AX (green) and mounted with DAPI (blue). Supplementary Figure S6: (A) MDA-MB-468, SQ20b or 231-H2N cells were exposed to IR (4 Gy) or sham-irradiated. After 1 or 4 h, cells were exposed for 2 h at 4{degree sign}C to increasing concentrations of 111In-EGF. To evaluate EGFR expression, the amount of membrane-bound 111In was determined. The number of EGFR/cell was unaltered by IR. (B) MDA-MB-468, SQ20b and 231-H2N cells were fixed, permeabilized and stained for EGFR (adapted from Cornelissen et al. J Nucl Med 2011; 52:776-783). Supplementary Figure S7. Proposed mechanism of action of 111In-anti-gH2AX-N-SS-E. (A) 111In-anti-gH2AX-N-SS-E binds to EGFR on the tumor cell membrane. (B) This binding triggers EGFR-mediated endocytosis and 111In-anti-gH2AX-N-SS-E becomes encapsulated in an endosome. (C) There, glutathione and reductive enzymes cleave the disulphide bond, to release 111In-anti-gH2AX-N, which (D) due to the NLS sequence, escapes from the endosome and localizes in the nucleus, through importin-NLS interaction. (E) gH2AX foci form following IR. (F) 111In-anti-gH2AX-N binds to gH2AX foci. (G) As 111In decays, it emits a shower of Auger electrons at the DSB site, which (H) causes more DSBs, and more gH2AX foci to be formed, eventually (I) leading to cell death. Supplementary Figure S8: 123I-labeled NLS shows a single peak on P4 SEC (black curve). When 123I-NLS was reacted with 0.8 mg/mL NaIO4 for 2 h there was no formation of higher molecular weight fragments (grey curve). However, after 24 h, there was evidence of dimerization (red curve).&lt;/p&gt;