Cibisatamab

PDOs were harvested with TrypLE Express and neutralised with DMEM/F12 Ham medium (Sigma Aldrich) with 10% FBS. Cells were filtered through a 70um filter, counted and re-suspended in phenol-red free RPMI medium (Thermo Fisher) supplemented with 10% FBS (Biosera), 1XGlutamax and 100units penicillin-streptomycin. On day 0, 5000 tumor cells per well of a 96 well-plate (Corning Special Optics Microplate) were plated. CD8 T cells were added on day 1 at the indicated effector to target (E:T) ratios with 20nM of cibisatamab or 20nM of the untargeted negative control antibody DP47-TCB (both provided by Roche). Tumor cells without CD8 T cells and without antibody were also included as controls. All conditions were plated in triplicates and at least 3 different healthy donors were tested on each of the 8 PDOs.

Human serum albumin (HSA) was used as plasma volume tracer and was labelled with ¹²⁵Iodine using the iodogen method, which was previously described in detail [22 (link),23 (link)]. Furthermore, 51Cr-EDTA (PerkinElmer Health Sciences B.V, Groningen, The Netherlands) served as an extracellular fluid tracer. For the biodistribution study, cibisatamab (20 mg/mL in 20 mM histidine/His-HCl, 240 mM sucrose, 10 mM methionine, 0.05% polysorbate 20 at pH 5.5) and CEACAM5-TCB (1.93 mg/mL in 20 mM Histidine, 140 mM NaCl at pH 6.0) (both provided by Roche) were also labelled with ¹²⁵Iodine. In order to avoid impact of the labelling procedure on the pharmacological activity, the two T-cell bispecific antibodies were labelled by an indirect labelling approach. After labelling, the retention of the biological activity was confirmed and potentially unbound ¹²⁵Iodine was removed prior to each injection. The labelling procedure and quality control is described in more detail in the supplementary material S1 and Figure S1 respectively.