Cetuximab

Imaging kinetics were measured using A431 cells cultured in a Millicell EZ 8‐Well Glass Slide (Millipore). The A431 cells were stained with 10 µg mL⁻¹ of SAFE‐IVM AF647‐labeled anti‐EGFR antibody (Cetuximab, Selleck Chemicals, A2000) for 10 min at RT. The stained cells were first imaged before addition of HK‐Tz. After the initial round of imaging, 40 µm of HK‐Tz was added to the cells for different lengths of time (5 s–30 min) to measure kinetics. After incubation, cells were washed three times with PBS and imaged.

In initial screens, PpL mutants and anti-CD3 (clone UTH1, BD bioscience 555329) antibodies were diluted into PBS pH7.6 such that the final concentrations were approximately 50 µM and 2 µM, respectively, and loaded into thin walled 200 µL polypropylene microtubes (PCR tubes). This mixture was then irradiated for 1 h under 365 nm light at an intensity of 6.4 mW/cm² from an LED source (M365LP1, Thor Labs) 14 cm away. Products were reduced using DTT solution (ThermoFisher) and separated on 4–12% BisTris PAGE gels (ThermoFisher) to observe photoconjugation. Full gel images are shown in Supplementary Fig. S2. Photocleavage was accomplished using the same irradiation setup. Photoconjugations to anti-FLAG antibody (anti-DYDDDDK clone 1557CT661.18.1, Lifespan Biosience LS‑C392574) or Cetuximab (Selleck Chemicals A2000) were done identically, with 100 µM PpL constructs that had been freshly purified. Photoconjugates were then purified from excess PpL using Amicon Ultra 50 kDa MWCO spin filter columns (Millipore-Sigma, UCFC505008). The unaltered antibody control conditions of our ELISA experiments, described below, validated Anti-FLAG binding to the FLAG peptide and Cetuximab binding to EGFR protein.

Chemicals (Cobimetinib, Afatinib, Erlotinib, Gedatosilib) were purchased from SelleckChem and antibodies (Cetuximab and Trastuzumab) were obtained from the Pharmacy Department of the Paul Strauss Cancer Center (CLCC, Nelly Etienne-Selloum). BHY and KYSE-510 cells at 70% confluency were grown with 10, 100, and 1000 ng/mL human epidermal growth factor (E9644-.2MG Sigma, Saint-Quentin-Fallavier, France) for 24 h.

Erastin (Selleck, Houston, TX, USA), RSL3 (Selleck, Houston, TX, USA), Sorafenib (Selleck, Houston, TX, USA), liproxstatin-1 (MCE, Princeton, NJ, USA), Cetuximab (Selleck, Houston, TX, USA), Gefitinib (Selleck, Houston, TX, USA), and staurosporine (GlpBio, Montclair, CA, USA) were dissolved in DMSO and kept at 80 °C.
HNSCC cells (CAL33, CNE-2, S18, S26, AMC-HN-8, U686) and N2A cells or primary neural stem cells (NSCs) were seeded into a 96-well plate with 8000 cells/wells, and DMSO, Erastin, RSL3, or Sorafenib were added into the medium the next day. Ferroptosis-inhibitors (liproxstatin-1) were added 2 h before Erastin and RSL3 or staurosporine. After culturing for 6 or 22 h, 10 μL of CCK-8 reagent (GlpBio, Montclair, CA, USA) was added and incubated at 37 °C and 5% CO₂ for 2 h. The absorbance was measured at 450 nm with a microplate analyzer.

In this study, we used an immunotoxin in which saporin was conjugated to cetuximab (Selleck.co.jp), hereafter referred to as IT‐cetuximab. IT‐cetuximab was prepared as follows: biotinylated cetuximab was purified by mixing cetuximab with EZ‐LINK sulfo‐NHS‐LC‐biotinylation kit (Thermo Fisher Scientific) at a 1:40 molar ratio using PD SpinTrap G‐25 (GE Healthcare Life Sciences). Next, biotinylated cetuximab and streptavidin‐saporin (Biotin‐Z Internalization Kit [KIT‐27‐Z]) (Advanced Targeting Systems) were mixed in equivalent amounts and allowed to react at room temperature for 30 min to obtain IT‐cetuximab.