Combining Cellular Immunization and Phage Display Screening Results in Novel, FcRI-Specific Antibodies

Antibodies that specifically bind to individual human fragment crystallizable gamma receptors (Fc gamma Rs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity Fc gamma Rs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor Fc gamma RI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human Fc gamma RI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of Fc gamma RI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with Fc gamma RI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new Fc gamma RI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing Fc gamma RI-restricted specificity.