PIKAMAB employs its proprietary Fc antibody engineering platform, Fc Walking™, to generate its MAb variants. As regards to ADCC antibody engineering, first we use our proprietary technology to stratify patients into three groups in each dimension (FcGR-3A: VV, VF, FF; and FcGR-2A: HH, HR, RR) to generate the 3×3 Matrix™, and through Fc Walking™, a set of related antibodies capable of modulating the responsiveness of a patient having an ADCC-treatable disease or disorder in each patient group of the matrix is generated.

Fc Walking is a codon-based, saturation mutagenesis (directed evolution) that integrates the 3×3 Matrix with the Fc:FcGR co-crystal structure information. Analogous to the conserved CDR stretches in the variable regions, we have identified similar conserved stretches in the Fc region. It is a bi-directional scanning saturation mutagenesis of approximately 5-10 residues, one residue at a time, on both sides of the “binding” motifs of the hFc regions namely lower hinge region, B/C loop, C’/E loop, and the F/G loop. There is no randomness involved in any of the steps as the location of change is predetermined. This method enables us to selectively control the critical parameters in antibody engineering: saturation mutagenesis, location of change, and extent of change. Briefly, all twenty natural amino acids are introduced for evaluation at each targeted position of interest, the variants are transiently expressed, and the affinity constants measured. This leads to generation of a family of related genes through codon-based mutagenesis. To further validate and optimize the variants, ex vivo assays are performed to measure ADCC. Thus, a small, ‘focused’ library is generated (instead of creating billions of random variants) from which optimized antibodies are selected, each one different from the parent gene by at least a single amino acid change at a pre-defined position.

This directed evolution approach significantly minimizes the risk of losing salient features during the optimization process because Fc Walking makes only the relevant amino acid substitutions necessary to create an optimized antibody.  This feature is particularly important for optimization of marketed MAbs because uncontrolled random changes necessitate the screening of enormous protein libraries, and can lead to undesirable results such as increased immunogenicity and toxicity.