A panel of PRLR-specific mAbs was generated using standard hybridoma technology following immunization with the extracellular domain (ECD, 25–234) of PRLR. Candidates were screened based on binding properties, and epitope, and candidates were humanized as IgG1 isotypes before conjugation to MMAE [19]. The lead mAb, h16f, was identified based on improved affinity, epitope binding properties and activity, and then engineered to include an S238C point mutation (S239C based on Kabat numbering system [20]] to permit site-specific DAR 2 conjugation to a PBD dimer (SGD-1882) via mc-Val-Ala linker as previously described [21].

Recombinant human, cynomolgus, and murine PRLR extracellular domains with a His-tag (huPRLR25–234, cyPRLR25–234 and muPRLR20–229) were expressed in and purified from HEK293 cells. Recombinant ECD of rat PRLR (residues 20–229 fused with poly-His tag at C-terminal), was purchased from Sino Biological Inc. and further purified by gel filtration using Superdex200 (GE Healthcare) in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA.

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