Directed evolution of super-secreted variants from phage-displayed human Interleukin-2

Abstract

Selection from a phage display library derived from human Interleukin-2 (IL-2) yielded mutated variants with greatly enhanced display levels of the functional cytokine on filamentous phages. Introduction of a single amino acid replacement selected that way (K35E) increased the secretion levels of IL-2-containing fusion proteins from human transfected host cells up to 20-fold. Super-secreted (K35E) IL-2/Fc is biologically active in vitro and in vivo, has anti-tumor activity and exhibits a remarkable reduction in its aggregation propensity- the major manufacturability issue limiting IL-2 usefulness up to now. Improvement of secretion was also shown for a panel of IL-2-engineered variants with altered receptor binding properties, including a selective agonist and a super agonist that kept their unique properties. Our findings will improve developability of the growing family of IL-2-derived immunotherapeutic agents and could have a broader impact on the engineering of structurally related four-alpha-helix bundle cytokines.

Affinity-matured variants derived from nimotuzumab keep the original fine specificity and exhibit superior biological activity

“…This finding implies that the primary driving force behind selection was target binding, and no other advantageous features like increased gene expression, improved protein periplasmic secretion and assembly on phage particles, and higher thermodynamic stability of the displayed protein. This result is in contrast with other examples of in vitro evolution where target-unrelated properties have been concurrently selected 24 .…”

“…While both nimotuzumab and K5 were predominantly monomeric (94.4 and 93.2% on average, respectively), K4 showed a major peak corresponding to 80.7% of the monomeric protein and almost 20% of large aggregates. There were no manufacturability advantages for the new antibodies as compared with the original one, as it has been observed for other molecules arising from directed evolution, which can have improved stability and solubility, and subsequently increased secretion levels 24 . In fact K4 seemed to be less produced and more aggregation-prone than nimotuzumab.…”

Baculovirus-free insect cell expression system for high yield antibody and antigen production

“…Supporting this hypothesis are the results of the expression of IL2. Here the point mutation, K35E 33 , leading to reduced aggregation and higher production yields in Expi293F cells, did not influence the yield in High Five cells, hinting that not aggregation is a bottleneck in High Five production. The observed lower aggregation of the antibodies is probably due to their simpler and more homogenous glycosylation pattern.…”

“…7b): Six antigens were produced in higher amounts in Expi293F cells, four in higher amounts in High Five cells and two reached approximately the same amount. Interestingly, a point mutation inserted in IL2-hFc, successfully increasing yields in Expi293F cells 33 (compare IL2-hFc and mutIL2hFc), did not influence the yield in High Five cells. Strikingly, all antigens were producible in High Five cells in a sufficient amount, whereas in Expi293F cells expression of www.nature.com/scientificreports/ hIL2R-mFc and S1-mFc failed, demonstrating that for some target proteins the insect cell expression system is better suited.…”

Building Personalized Cancer Therapeutics through Multi-Omics Assays and Bacteriophage-Eukaryotic Cell Interactions

“…Phage display is based on the fact that an encapsulated library of genotypes can be directly associated with the presentation of a library of molecules on the phage surface. Phage display has been used in a variety of applications, including epitope mapping—where a library of peptide expressing phage is used to interact with a specific antibody, therefore to pinpoint the specific epitope sequence the antibody is interacting with [ 112 ]; ligand identification for receptors—similar to antibody mapping, peptides interacting with receptors can be identified [ 113 ]; protein-protein interactions—where phage can present large proteins that are potentially interacting with a known binding partner, therefore to identify the unknown binding partners and to study the mechanism of interactions [ 114 ]; directed evolution of proteins—mutations conferring binding advantages between two proteins can be studied using a phage display library containing these variants [ 115 ]; drug discovery—peptides or ligands that can interact with drug targets can be presented through phage display [ 116 ]; and antibody screening—where a large library of antibody-displaying phage can be screened for the best antibodies that can interact with the target antigens [ 117 ].…”