Deferoxamine maleimide

To label recPrP with zirconium-89, full length recombinant mouse PrP (23–230) generated in E.coli was first conjugated to deferoxamine-maleimide (Macrocyclics) to produce deferoxamine-conjugated PrP (DFO-recPrP). To ensure that the linker was not conjugated to the lysine-rich heparin binding domain, recPrP was bound to heparin sepharose beads to block the heparin binding sites. To block the binding sites, Heparin Sepharose 6 Fast Flow beads (1 ml) (Healthcare Life Sciences) were loaded into disposable Bio-Spin chromatography columns (Bio-Rad) and packed with 2 ml of equilibration buffer (0.15 M NaCl in 25 mM HEPES, pH 7.4). RecPrP (200 μg) was mixed with 1 ml of equilibration buffer and applied onto the columns. The columns were washed with 3 ml of equilibration buffer. The beads were then transferred to a 1.5 ml eppendorf polypropylene tube and incubated with 200 μl of deferoxamine-maleimide for 24 hours at room temperature with rotation. The bead slurry was transferred to a chromatography column and the recPrP conjugation was stopped after 24 hours by adding 600 μl PBS with 0.1 M glycine (pH 7). The beads were washed with 2 ml equilibration buffer, and the conjugated recPrP (DFO-recPrP) was eluted with 1 ml of elution buffer (0.7 M NaCl, 25 mM HEPES, pH 7.2). DFO-recPrP was concentrated using Zeba spin desalting columns.

Deferoxamine-maleimide was from Macrocyclics (Dallas, TX), and matrigel was purchased from Becton Dickenson Biosciences (Bedford, MA). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and phorbol-12-myristate-13-acetate (PMA) was from Sigma-Aldrich (St. Louis, MO). ⁸⁹Zr-oxalate was from the Korea Atomic Energy Research Institute (Daejeon, Korea). Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH). Among antibodies for Western blotting, mouse IgG against β-actin was from Santa Cruz Biotechnology (Dallas, Texas; #sc-47778) and rabbit IgG against CD25 was from Abcam (Cambridge, MA; #ab231441). HRP-conjugated secondary anti-mouse and anti-rabbit IgG was from Cell Signaling Technologies (Beverly, MA).
Human anaplastic large cell lymphoma SUDHL-1 cells and EL4 mouse T lymphoma cells were from the American Type Culture Collection and was tested negative for mycoplasma and authenticated by the Institutional Research Service. Cells were maintained in 5% CO₂ at 37°C in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/ml penicillin-streptomycin. Cells were sub-cultured twice a week and used when confluence was 80%.

Prior to conjugation of the purified GaletuzuFab-PAS200-Cys with Dfo or Cy7 using appropriate derivatives carrying a maleimide group, the single unpaired Cys residue was liberated from mixed disulfides with other thiol compounds by mild reduction with a tenfold molar amount of dithiothreitol (DTT) in PBS at 20 °C, followed by separation of reagents and side products on a PD10 column (Sigma-Aldrich, Taufkirchen, Germany). To restore any intramolecular disulfide bonds that may have become affected by this procedure, the Fab was subsequently incubated with a 20-fold molar amount of (l)-dehydroascorbic acid (dhAA; Sigma-Aldrich) for 3 h at 20 °C. After another salt exchange on a PD10 column to 150 mM ammonium acetate (99.999% trace metals basis; Sigma-Aldrich), the Fab (2 mg/ml) was incubated with the fivefold molar amount of cyanine-7-maleimide (Lumiprobe, Cockeysville, MD, USA) or deferoxamine-maleimide (Macrocyclics, Plano, TX, USA) over night at 4 °C. Excess reagent was again removed on a PD10 column using 150 mM ammonium acetate as running buffer, and the composition of each conjugate was finally checked by ESI–MS.