Studying SARS-CoV-2 interactions using phage-displayed receptor binding domain as a model protein.

SARS-CoV-2 receptor binding domain (RBD) mediates viral entry into human cells through its interaction with angiotensin converting enzyme 2 (ACE2). Most neutralizing antibodies elicited by infection or vaccination target this domain. Such a functional relevance, together with large RBD sequence variability arising during viral spreading, point to the need of exploring the complex landscape of interactions between RBD-derived variants, ACE2 and antibodies. The current work was aimed at developing a simple platform to do so. Biologically active and antigenic Wuhan-Hu-1 RBD, as well as mutated RBD variants found in nature, were successfully displayed on filamentous phages. Mutational scanning confirmed the global plasticity of the receptor binding motif within RBD, highlighted residues playing a critical role in receptor binding, and identified mutations strengthening the interaction. The ability of vaccine-induced antibodies to inhibit ACE2 binding of many mutated RBD variants, albeit at different extents, was shown. Amino acid replacements which could compromise such inhibitory potential were underscored. The expansion of our approach could be the starting point for a large-scale phage-based exploration of diversity within RBD of SARS-CoV-2 and related coronaviruses, useful to understand structure-function relationships, to engineer RBD proteins, and to anticipate changes to watch during viral evolution.

Safety and immunogenicity of anti-SARS CoV-2 vaccine SOBERANA 02 in homologous or heterologous scheme: Open label phase I and phase IIa clinical trials.

BACKGROUND: SOBERANA 02 is a COVID-19 vaccine based on SARS-CoV-2 recombinant RBD conjugated to tetanus toxoid (TT). SOBERANA Plus antigen is dimeric-RBD. Here we report safety and immunogenicity from phase I and IIa clinical trials using two-doses of SOBERANA 02 and three-doses (homologous) or heterologous (with SOBERANA Plus) protocols. METHOD: We performed an open-label, sequential and adaptive phase I to evaluate safety and explore the immunogenicity of SOBERANA 02 in two formulations (15 or 25 µg RBD-conjugated to 20 µg of TT) in 40 subjects, 19-59-years-old. Phase IIa was open-label including 100 volunteers 19-80-years, receiving two doses of SOBERANA 02-25 µg. In both trials, half of volunteers were selected to receive a third dose of the corresponding SOBERANA 02 and half received a heterologous dose of SOBERANA Plus. Primary outcome was safety. The secondary outcome was immunogenicity evaluated by anti-RBD IgG ELISA, molecular neutralization of RBD:hACE2 interaction, live-virus-neutralization and specific T-cells response. RESULTS: The most frequent adverse event (AE) was local pain, other AEs had frequencies ≤ 5%. No serious related-AEs were reported. Phase IIa confirmed the safety in 60 to 80-years-old subjects. In phase-I SOBERANA 02-25 µg elicited higher immune response than SOBERANA 02-15 µg and progressed to phase IIa. Phase IIa results confirmed the immunogenicity of SOBERANA 02-25 µg even in 60-80-years. Two doses of SOBERANA02-25 µg elicited an immune response similar to that of the Cuban Convalescent Serum Panel and it was higher after the homologous and heterologous third doses. The heterologous scheme showed a higher immunological response. Anti-RBD IgG neutralized the delta variant in molecular assay, with a 2.5-fold reduction compared to D614G neutralization. CONCLUSIONS: SOBERANA 02 was safe and immunogenic in persons aged 19-80 years, eliciting neutralizing antibodies and specific T-cell response. Highest immune responses were obtained in the heterologous three doses protocol. TRIAL REGISTRY: https://rpcec.sld.cu/trials/RPCEC00000340, https://rpcec.sld.cu/trials/RPCEC00000347.

A COVID-19 vaccine candidate composed of the SARS-CoV-2 RBD dimer and Neisseria meningitidis outer membrane vesicles.

SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host's cellular receptor. Vaccines seek to block this interaction by eliciting neutralizing antibodies, most of which are directed toward the RBD. Many protein subunit vaccines require powerful adjuvants to generate a potent antibody response. Here, we report on the use of a SARS-CoV-2 dimeric recombinant RBD combined with Neisseria meningitidis outer membrane vesicles (OMVs), adsorbed on alum, as a promising COVID-19 vaccine candidate. This formulation induces a potent and neutralizing immune response in laboratory animals, which is higher than that of the dimeric RBD alone adsorbed on alum. Sera of people vaccinated with this vaccine candidate, named Soberana01, show a high inhibition level of the RBD-ACE2 interaction using RBD mutants corresponding to SARS-CoV-2 variants of concern and wild-type expressed using the phage display technology. To our knowledge, this is the first time that the immunostimulation effect of N. meningitidis OMVs is evaluated in vaccine candidates against SARS-CoV-2.

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.

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

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.

High throughput functional epitope mapping: revisiting phage display platform to scan target antigen surface.

Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor.

Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.