FcRn interaction characterization of the BsAbs
BsAb-1 and BsAb-2 were evaluated for their cynomolgus monkey FcRn (cFcRn) binding properties using two previously reported assays.24, 25 First, the cFcRn binding affinity (ie KD at pH 6 and 7.4) was determined using SPR. No significant difference in cFcRn binding between the two molecules was observed at pH 6. The binding affinity (KD) of the BsAb-1 and BsAb-2 for cFcRn at pH 6.0 were ~105 and ~115 nM, respectively (Table 2). No direct binding to cFcRn at neutral pH was detected for either of the BsAbs (Table 2).
In the second assay, the ability of the BsAbs to dissociate from the receptor once bound was measured using an ELISA in a pH-dependent manner (Table 2).24 In this format, we form complexes of the each BsAb with cFcRn at acidic pH (pH 6) and measure the amount of each molecule which remains bound to cFcRn once the complex is exposed to neutral pH (pH 7.4) as a surrogate of cFcRn intracellular binding and extracellular release; thus, the higher the percent of molecule bound to cFcRn the less efficient the release from the receptor at neutral pH. BsAb-1 and BsAb-2 show ~54% and ~5%, respectively, remain bound to cynomolgus monkey FcRn once the complex is exposed to neutral pH (Table 2). Intrigued by this finding, we evaluated mAb-1 which is the mAb component of BsAb-1 and found mAb-1 showing ~3% remains bound to cynomolgus monkey FcRn under the same conditions (data not shown). Evaluation of the human FcRn pH-dependent release profile for each BsAb showed similar findings to cFcRn (data not shown).