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).