No abstract

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-19 1,2 . A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-19 3 . Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.The COVID-19 pandemic has prompted substantial efforts to develop effective countermeasures against SARS-CoV-2. Preclinical data and phase-III clinical studies indicate that monoclonal antibodies could be effectively deployed for prevention or treatment during the viral symptoms phase of the disease 1,2 . Cocktails of two or more monoclonal antibodies are preferred over a single antibody as these cocktails result in increased efficacy and the prevention of viral escape. However, this approach requires increased manufacturing costs and volumes, which are problematic at a time when the supply chain is under pressure to meet the high demand for COVID-19 therapeutic agents, vaccines and biologics in general 4 . Cocktails also complicate formulation 5,6 and hinder strategies such as antibody delivery by viral vectors or by nonvectored nucleic acids 7,8 . One alternative is to use multispecific antibodies, which have the advantages of cocktails and single-molecule strategies.To this end, we used structural information 9 and computational simulations to design bispecific antibodies that would simultaneously bind to (i) independent sites on the same RBD and (ii) distinct RBDs on a spike (S) trimer. We evaluated several designs using atomistic molecular dynamics simulations, and produced four constructs: of these, CoV-X2 was the most potent neutralizer of SARS-CoV-2 pseudovirus, and had a half-maximal inhibitory concentration (IC 50 ) of 0.04 nM (5.8 ng ml −1 ) (Extended Data Fig. 1). CoV-X2 is a human-derived IgG1-like bispecific antibody in the CrossMAb format 10 that is the result of the combination of the Fragment antigen binding (Fab) of the monoclonal antibodies C121 and C135, which are two potent neutralizers of SARS-CoV-2 3 . Structural predictions showed that CoV-X2-but not its parental monoclonal antibodies-can bind bivalently to all RBD conformations on the S trimer, which prevents the binding of ACE2 receptor 11 (Fig. 1a, Extended Data Fig. 2).CoV-X2 bou...

From February to April 2020, Lombardy (Italy) reported the highest numbers of SARS-CoV-2 cases worldwide. By analyzing 346 whole SARS-CoV-2 genomes, we demonstrate the presence of seven viral lineages in Lombardy, frequently sustained by local transmission chains and at least two likely to have originated in Italy. Six single nucleotide polymorphisms (five of them non-synonymous) characterized the SARS-CoV-2 sequences, none of them affecting N-glycosylation sites. The seven lineages, and the presence of local transmission clusters within three of them, revealed that sustained community transmission was underway before the first COVID-19 case had been detected in Lombardy.