Abstract

We develop new technologies for surface display of full-length IgG antibody, VHH nanobody and antibody fragments (Fab, scFv) by using yeast P. pastoris, wherein the N-glycosylation pathway is humanized. These technologies integrate mammalian cell quality control with yeast display’s high-throughput screen into one platform. The quality control in antibody screening will address multiple developability issues in the early stages of lead discovery. Here we present several case studies of antibody engineering by using our yeast display technologies. Antibodies can be highly toxic, because they target both tumor tissue and normal tissue. Since the extracellular pH in most solid tumors is lower than that of normal tissues, we applied our yeast display platform to engineer pH-dependent antibody with selective binding in the acidic tumor microenvironment aiming at improving antibody target selectivity and reducing off-target antibody toxicity. The common light chain bispecific antibody is an elegant technology that simplifies the production of IgG–like bispecific antibody. We used our yeast display platform to perform a high-throughput screening of light chain library to isolate common chains for two different original antibodies. It is important to determine antibody cross-reactivity to ensure that is specific enough for the intended use. Antibodies raised against a specific human antigen may not cross-react with nonhuman antigen, which can affect the choice and efficacy of pre-clinical animal models. On the other hand, cross-reactivity can cause the antibody to bind to non-specific antigens, leading to false positive or other errors in the assay. We used our yeast display platform and co-selection strategy to select nanobody cross-reactivity from an immune library raised against a human antigen. A high-throughput flow cytometry assay was used to confirm the species cross-reactivity of selected nanobodies. A group of nanobodies have specific binding to human antigen but no cross-reactivity to mouse antigen. Another group of nanobodies have cross-reactivity to both human and mouse antigens. Our yeast display platform and co-selection strategy can broadly apply to screen antibody library for species cross-reactivity.
Keywords: Antibody engineering, Yeast Display, pH-dependent Antibody, Bispecific Antibody, Species cross-reactivity,