Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy

Abstract

As of June 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global emergency and effective therapeutic interventions for the treatment and prevention of coronavirus disease 2019 (COVID-19) are urgently needed. SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) represent a promising approach to COVID-19 therapy. However, the recently described accumulating mutations in the SARS-CoV-2 spike protein are challenging the efficacy of approved and investigational mAbs, whose widespread use is also hampered by their significant costs and possible side effects, including Antibody-Dependent Enhancement (ADE). Here we describe a cluster of SARS-CoV-2 neutralizing human single chain variable fragment antibodies, identified by phage display, sharing a common VH CDR3 sequence. Phage libraries were built by amplifying variable domains of immunoglobulin genes from cDNA derived from lymphocytes of COVID-19 convalescent subjects living in Bergamo, Italy. The scFv76-cluster antibodies (scFv76-cl Abs) exhibit high affinity for the spike receptor binding domain (RBD) of Wuhan strain and emerging variants, leading to inhibition of RBD/human ACE2 interaction. The antigenic epitope recognized by scFv76 was mapped in the receptor binding motif (RBM) of RBD at residues L455, F456, Y473, N487 and Y489. None of these residues has been to date listed among the RBD mutations of SARS-CoV-2 variants of concern (VOCs), suggesting an important role of such epitope in viral infectivity. Treatment with scFv76-cl Abs is effective against SARS-CoV-2, as determined by in vitro experiments of viral infection, replication, cytopathogenicity and spike-mediated syncytia formation. Moreover, their intranasal administration is shown to counteract infection in two independent animal models. Overall, the biochemical and biological characteristics of scFv76-cl Abs are compatible with their clinical use for COVID-19 therapy by intranasal or aerosol administration. To our knowledge, this is the first example of promising human anti-SARS-CoV-2 scFv antibodies as drug candidates for COVID-19 therapy.

Spike mutation resilient scFv76 antibody counteracts SARS-CoV-2 lung damage upon aerosol delivery

“…The scFv76 paratope comprises residues of all three heavy chain complementarity determining regions (CDRs), and two from the light chain CDRs ( Table 3 and Figure 5B ). Conversely, the recognition site lies on the RBD receptor-binding ridge and surrounding areas ( Figure 5C ), in full agreement with alanine scanning analysis previously reported ( 12 ), whereby RBD L455A, F456A, Y473A, N487A and Y489A mutations strongly reduced scFv76 binding. Of note, F456 is located within the deep groove created between CDRH1 and CDRH2, on one side, and CDRH3 and CDRL3 on the other.…”

“…Binding affinity of scFv76 to the Omicron spikes was then tested by Surface Plasmon Resonance (SPR) showing K D of 6.3 and 14.5 nM for BA.1 and BA.2, respectively ( Table 2 ). Neutralizing activity against SARS-CoV-2-S Omicron BA.1 and BA.2 pseudotyped viruses was also exhibited by scFv76, but not by scFv5 (an anti-RBD antibody previously shown to be devoid of neutralizing activity and used as negative control) ( 12 ), with an IC50 value of 2.84 and 2.47 nM, respectively ( Figure 1A ).…”

“…The scFv76 antibody was previously described to be able to neutralize the SARS-CoV-2 Alpha, Beta, Gamma and Delta viral variants both in vitro and in animal models ( 12 ). To evaluate its reactivity with the recently emerged Omicron variants, the ability to compete the binding of the Omicron BA.1 and BA.2 spikes to human ACE2 was tested by ELISA.…”

“…We previously established the biochemical suitability of scFv76 to aerosol delivery by a mesh nebulizer ( 12 ). To test pharmacological efficacy of the aerosolized antibody, pneumonia infection was established in transgenic hACE2 mice by intranasal challenge with 1×10 5 TCID50 SARS-CoV-2 (Strain Delta B.1.617.2).…”

“…We recently described a cluster of human anti-SARS-CoV-2 antibodies in the format of single-chain variable fragment (scFv) able to neutralize viral variants both in vitro and in animal models ( 12 ). We also showed that such antibody format is suitable for intra-nasal or aerosol formulations that might be useful for the topical treatment of upper and lower respiratory tract SARS-CoV-2 infections ( 12 ).…”