Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles.

Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.

A cell-based ELISA as surrogate of virus neutralization assay for RBD SARS-CoV-2 specific antibodies.

SARS-CoV-2, the cause of the COVID-19 pandemic, has provoked a global crisis and death of millions of people. Several serological assays to determine the quality of the immune response against SARS-CoV-2 and the efficacy of vaccines have been developed, among them the gold standard conventional virus neutralization assays. However, these tests are time consuming, require biosafety level 3 (BSL3), and are low throughput and expensive. This has motivated the development of alternative methods, including molecular inhibition assays. Herein, we present a safe cell-based ELISA-virus neutralization test (cbE-VNT) as a surrogate for the conventional viral neutralization assays that detects the inhibition of SARS-CoV-2 RBD binding to ACE2-bearing cells independently of species. Our test shows a very good correlation with the conventional and molecular neutralization assays and achieves 100% specificity and 95% sensitivity. cbE-VNT is cost-effective, fast and enables a large-scale serological evaluation that can be performed in a BSL2 laboratory, allowing its use in pre-clinical and clinical investigations.

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

Nimotuzumab is a humanized monoclonal antibody against the Epidermal Growth Factor Receptor with a long history of therapeutic use, recognizing an epitope different from the ones targeted by other antibodies against the same antigen. It is also distinguished by much less toxicity resulting in a better safety profile, which has been attributed to its lower affinity compared to these other antibodies. Nevertheless, the ideal affinity window for optimizing the balance between anti-tumor activity and toxic effects has not been determined. In the current work, the paratope of the phage-displayed nimotuzumab Fab fragment was evolved in vitro to obtain affinity-matured variants. Soft-randomization of heavy chain variable region CDRs and phage selection resulted in mutated variants with improved binding ability. Two recombinant antibodies were constructed using these variable regions, which kept the original fine epitope specificity and showed moderate affinity increases against the target (3-4-fold). Such differences were translated into a greatly enhanced inhibitory capacity upon ligand-induced receptor phosphorylation on tumor cells. The new antibodies, named K4 and K5, are valuable tools to explore the role of affinity in nimotuzumab biological properties, and could be used for applications requiring a fine-tuning of the balance between binding to tumor cells and healthy tissues.