Protein engineering for Improved Thermostability of a Bispecific IL2-mimic Antibody by phage display
Peter Brauer, Hwee Ching Tan, Siok Ping Yeo and Cheng-I Wang
Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore
Interleukin -2 (IL-2) is an immunostimulatory cytokine that acts as a mitogen for T cells and natural killer (NK) cells. There has been increasing interest in using IL-2 as a therapeutic agent in cancers and infectious diseases. However, to date, it has not been successfully applied in a clinical setting due to several reasons. IL-2 signals via the IL-2 receptor complex (IL-2R), comprising three subunits: IL-2Rα (CD25), IL-2Rβ (CD122) and IL-2Rγ (CD132, common gamma chain, γc). IL-2Rα binds IL-2, presenting the cytokine to both IL-2Rβ and γc, which formed the high affinity IL-2R complex, resulting in downstream signaling. In the absence of IL-2Rα, the complex has low affinity for IL-2; hence IL-2Rα negative cells are less IL-2 stimulated. The three subunits are differentially expressed on T cell subsets, with naїve and regulatory T cells (Treg) having high expression of IL-2Rα, and relatively low expression of IL-2Rβ and γc. IL-2 therapy stimulates Treg, which interfere with effector cell immune function. Previous work done on the design of an agonistic bispecific antibody that bypasses IL-2Rα-dependent Treg stimulation and toxicity had been successfully demonstrated in in vitro assays. However, this bispecific antibody clone has very low potency, possibly due to its poor thermal stability. Here, we describe a methodology combining random mutagenesis by error-prone PCR (epPCR) and Megaprimer PCR of Whole Plasmid (MEGAWHOP) and stringent selection by a tailored biopanning protocol (phage display) to select for thermostable bispecific antibody clones. This effort resulted in a panel of thermostable bispecific clones with improved binding affinities for the target antigens. We further characterized the mutations individually to determine which ones contributed to the thermal stabilization of the molecule.
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