Preparation of targeting conjugates

Anti-EGFR ZEGFR:2377 affibody molecule having a single C-terminal cysteine was produced as previously described (38).

For labelling with ⁸⁹Zr, ZEGFR:2377 was conjugated to a maleimido derivative of deferoxamine (DFO) chelator (Macrocyclics, Dallas, TX, USA). To reduce spontaneously formed intermolecular disulphide bonds, affibody molecules were treated with dithiothreitol (DTT; E. Merck, Darmstadt, Germany). Affibody molecules (2 ml, 2.3 mg/ml in PBS) were mixed with 100 μl 1 M Tris-HCl buffer, pH 8.0, and 63 μl DTT solution (0.5 M in water). The mixture was incubated at 40°C for 30 min. The reduced affibody molecules were then purified and the buffer was changed using a disposable PD-10 column (GE Healthcare, Uppsala, Sweden) pre-equilibrated with 0.2 M ammonium acetate, pH 5.5. The DFO-to-ZEGFR:2377 ratio was optimized to obtain conjugate in high yield. To do this, a 2-, 3-, 5- and 8-fold molar excess of DFO (0.0337 μmole/μl DMSO) was added to the ZEGFR:2377 (0.5 mg in 0.72 ml 0.1 M ammonium acetate buffer, pH 5.5) under gentle shaking, and incubated for 30 min at 40°C. Unconjugated molecules and excess chelators were separated from the DFO-conjugated affibody molecules on a semi-preparative RP-HPLC column (Zorbax 300SB-C18 9.4×250 mm, 5 μm particle size; Agilent Technologies, Palo Alto, CA, USA) using a gradient from 35–60% B for 18 min at a flow rate of 0.5 ml/min (A: 0.1% trifluoroacetic acid in water, B: 0.1% trifluoroacetic acid in acetonitrile). The analysis was performed using high-performance liquid chromatography and on-line mass spectrometry (HPLC-MS) using an Agilent 1100 LC/MSD system equipped with electrospray ionization and single quadrupole (Agilent Technologies). The analysis was performed using a Zorbax 300SB-C18 2.1×150 mm, 3.5 μm column. Agilent ChemStation Rev. B.02.01 software (Agilent Technologies) was used for analysis and evaluation of HPLC data. The purified conjugate (further designated as DFO-ZEGFR:2377) was freeze-dried.

Cetuximab was conjugated with p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS) as previously described (39). The conjugate was purified using a NAP-5 size-exclusion column equilibrated with PBS.