P scn bn dota dota

The antibodies HH1 (anti-CD37, Nordic Nanovector ASA, Oslo, Norway), Rituximab (anti-CD20, Roche, Pharma Schweiz, Basel, Switzerland) and the murine non-specific isotype control antibody (IgG₁) (MAB002, R&D Systems Inc., Minneapolis, USA) were labeled with the chelator p-SCN-Bn-DOTA (DOTA, Macrocyclics, TX, USA) and subsequently labeled with ¹⁷⁷Lu (ITG, Garching, Germany) as previously described [26 (link)]. Labeling with ¹²⁵I (Hartmann Analytic, Braunschweig, Germany) was performed using iodogen tubes (Pierce, Rockford, IL, USA) as described previously [23 (link)].
The immunoreactivity (IRF) of all the tumor specific RICs was verified using a modified Lindmo method [27 (link)] with one cell concentration of 75 million cells/ml. The IRF of all specific RICs was between 57% and 76%.

Reagents and solvents: All chemicals and solvents were purchased from commercial suppliers such as Sigma-Aldrich (Bornem, Belgium), Fluka (Bornem, Belgium), Fisher (Doornik, Belgium) and Acros Organics (Geel, Belgium) and were used without further purification. p-SCN-Bn-DOTA (DOTA) was purchased from Macrocyclics, Inc (Plano, Texas, USA). Details regarding the synthesis of 3p-C-NETA-(tBu)-oxa-butanoic acid 16 (link) and 3p-C-NETA-TATE 17 can be found in the supplementary data.

Humanized 5A10 (hu5A10) (Innovagen AB, Lund and Diaprost) was conjugated with either CHX-A″-DTPA (Macrocyclics, Plano, TX, USA) or p-SCN-Bn-DOTA (DOTA) (Macrocyclics) at a 3:1, 6:1, or 12:1 chelator-to-antibody molar ratio. The conjugates were stored at −8 °C and used/labeled within the same day of conjugation (see Appendix A).
Labeling efficiency of the radiolabeled conjugates was evaluated using a previously established thin-layer chromatography protocol, which utilized a phosphor imager system and Optiquant software (all details are given in the Appendix A).
The average number of chelators conjugated to each mAb was determined according to Mears et al. [32 (link)]. In brief, the immunoconjugate was mixed with a ¹¹¹InCl₃/^natInCl₃ solution consisting of known concentrations of non-radioactive (Sigma-Aldrich, Saint-Louis, MO, USA) and radioactive (¹¹¹In) indium. The radioactive yield was, together with the assumption of a 1:1 interaction between indium atoms and chelators, utilized for calculating the number chelates attached to each mAb.