125i stock solution

All four affibodies were radiolabeled by direct iodination using the chloramine T method (42 (link)). Briefly, 20–40 μg affibody was mixed with ¹²⁵I stock solution (Perkin-Elmer Inc Waltham, MA, USA). Thereafter, 5 μg Chloramine-T (Sigma Aldrich, Stockholm, Sweden) in PBS was added to a final volume of 110 μl. The reaction was incubated for 90 s at room temperature, and quenched with 10 μg sodium metabisulfite (Sigma Aldrich). The product was diluted to 500 μl with PBS, and separated from free-iodine in a PBS-equilibrated NAP-5 size exclusion column (Cytiva, Uppsala, Sweden). The product was eluted with a total volume of 1 ml PBS.
Pierce Pre-Coated Iodination Tubes (ThermoFischer, Rockford, IL, USA) was used as an alternative to the Chloramine T method to achieve milder reaction conditions (43 (link)). First, 1 ml PBS was used to wet the iodination tube. ¹²⁵I stock solution (Perkin-Elmer) was added together with PBS to the iodination tube to a total volume of 40 μl. The tube was incubated for 6 min on a shaker at room temperature to activate the iodine. 10–20 μg affibody was prepared in Protein LoBind tubes (Eppendorf). Thereafter, 20 μl activated iodine was added to each affibody, followed by a 10 min incubation at room temperature on a shaker (600 rpm). After the incubation, the product was diluted with PBS to 500 μl and purified as described above.

The scFvs variants were labelled with iodine-125 (¹²⁵I) using Chloramine-T as described previously^{92 (link)}. In short, 400 nM of the three scFvs were mixed with ¹²⁵I stock solution (Perkin Elmer Inc, Waltham) and 1 mg/mL of Chloramine-T (Sigma Aldrich) in PBS. After 90 s of incubation, the reaction was stopped with 1 mg/mL sodium meta-bisulphite (Sigma 08982). The radio-labeled scFvs were then purified from unbound and free ¹²⁵I by using NAP columns (VWR 17-0853-02) and eluted in PBS. The radiolabeling procedures were done 1–2 h before the ex vivo studies. The yield of the labeling process varied between the different studies, due to inherent variation of the method, however this was accounted for in the dose calculations. The yield was approximately 22.8 MBq/nmol for the in vivo 96 h plasma stability study, 35 MBq/nmol for the 2 h and 48 h ex vivo studies, approximately 12 MBq/nmol for the NTE/CD ex vivo study (also 2 h) and approximately 3.5 MBq/nmol for the 24 h therapeutic ex vivo study.

The two bispecific antibodies, mAb3D6-scFv8D3 and di-scFv3D6-8D3, were radiolabelled by direct iodination with iodine-125 (¹²⁵I) using the Chloramine-T method [32 (link)]. In short, mAb3D6-scFv8D3, or di-scFv3D6-8D3, was mixed with ¹²⁵I stock solution (Perkin-Elmer Inc Waltham, MA, USA) and 5 μg Chloramine-T (Sigma Aldrich, Stockholm, Sweden) in PBS to a final volume of 110 μl. The reaction was incubated for 90 s at room temperature, and then quenched with 10 μg sodium metabisulfite (Sigma Aldrich). The product was diluted to 500 μl with PBS, and separated from free-iodine and low molecular weight products with a NAP-5 size exclusion column (GE Healthcare, Uppsala, Sweden) with a total elution volume of 1 ml PBS.