Conjugation of scFv78-Fc with p-SCN-Bn-CHX-A”-DTPA and Radiolabeling with Indium-111

The scFv78-Fc fusion protein targeting endosialin/TEM1 was obtained as previously described [33]. A solution of scFv78-Fc (1.19 mg/ml, 960 μl) was concentrated to ca. 200 μl by ultrafiltration (Amicon Ultra, 0.5 ml, 50 kD) in two successive 450 μl additions and spins (2 × 4 min at 9000 g). Subsequently, 67 μl (10 eq.) of a freshly made solution of 1.0 mg of p-SCN-Bn-CHX-A”-DTPA · 3 HCl (Macrocyclics, Cat-N° B-355) in dimethyl sulfoxide (DMSO) (50 μl) and carbonate-bicarbonate (CBC) buffer (0.2 M, pH 9.1, 1 ml) were added to the concentrated scFv78-Fc solution and incubated for 1 h at 42 °C. Then, dilution with 500 μl of NaCl 0.9 % was effected, and the excess chelator was removed by ultrafiltration (3 × 6 min at 9000 g). The ca. 40 μl solution was diluted with 190 μl of NaCl 0.9 % to give a final concentration of 5 mg/ml CHX-DTPA-scFv78-Fc.

Mass spectrometry (MS) was performed using a Q Exactive HF (Thermo Fisher) operating in the high mass range in order to quantify the mean number of chelators per antibody. Samples were diluted to 1 μg/μl in NH₄OAc (pH 7, 50 mM) and injected in a MAbPac™ SEC-1 column (Thermo Fisher Scientific) using NH₄OAc (pH 7, 50 mM) at 0.3 ml/min as mobile phase. MS spectra were acquired in the 300–8000 m/z at a resolution set to 15 K. The MS spectra were deconvoluted using Protein Deconvolution (Thermo Fisher Scientific). The analysis showed a distribution between 3 and 6 CHX-DTPA chelators per scFv78-Fc, with a mean value of about 4 chelators per antibody (Fig. 1).

Mass spectrometry of scFv78-Fc (bottom) and of CHX-DTPA-scFv78-Fc (top). The delta of m/z 2449 between mean peaks suggests an average of 4 CHX-DTPA per antibody with a distribution between 3 and 6.

[¹¹¹In]CHX-DTPA-scFv78-Fc was obtained by adding a commercially available [¹¹¹In]indium chloride solution (200 MBq, Mallinckrodt) to a mixture of NH₄OAc (pH 5.4, 0.4 M, 100 μl) and CHX-DTPA-scFv78-Fc (50 μl, 250 μg). The radiolabeling yield was assessed by instant thin layer chromatography on silica gel (iTLC-SG) with citrate buffer pH 4.6.