Generation of Neutralizing Monoclonal Antibodies Against SARS-CoV-2 for Prevention and Therapy in Patients with COVID-19

National Institute Of Allergy And Infectious Diseases

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Abstract

Potent monoclonal antibodies neutralize Omicron sublineages and other SARS-CoV-2 variants The RNA nature and broad circulation of SARS-CoV-2 enable the accumulation of mutations leading to the continuous emergence of variants with increased transmissibility or pathogenicity as well as resistance to monoclonal antibodies (mAbs) and vaccine-elicited antibodies, highlighting the need for effective therapeutic and preventive measures with a broad spectrum of action. A. Generation and characterization of anti-SARS-CoV-2 monoclonal antibodies using phage-display libraries from COVID-19 convalescent patients. Screening of more than 120 convalescent patients from COVID-19, recruited at the NIH Blood Bank, identified 12 convalescent COVID-19 patients with high neutralizing serum antibody titers against SARS-CoV-2. Peripheral blood samples were collected from these 12 individuals, and total RNA was extracted and used for the construction of four phage-display Fab libraries derived from either a single donor or multiple donors combined, each consisting of a huge number of individual clones. Three sequential cycles of panning were carried out to enrich for specific clones using a stabilized trimeric spike protein (S-2P) derived from the original SARS-CoV-2 strain, Wuhan-Hu-1. Subsequent screening of 672 individual clones by ELISA resulted in the identification of 538 clones that specifically bound to the S-2P protein. DNA sequencing identified 18 unique clones with distinct sequences. These clones were then subcloned and expressed both as soluble Fabs and as complete IgG1 antibodies. The binding specificity of the cloned IgGs was confirmed by ELISA. The binding affinity of the 18 Fabs for the soluble S-2P trimer was further assessed by surface plasmon resonance (SPR), which documented that 12 out of the 18 exhibited high-affinity binding with equilibrium dissociation constants (KD) in the picomolar range for 10 and in the low nanomolar range for 2. B. Potent neutralization of diverse SARS-CoV-2 variants of concern. The neutralizing activity of our 18 mAbs was evaluated using a pseudotype virus neutralization assay. Eleven showed potent neutralizing activity against the original SARS-CoV-2 strain (WA-1), 7 with a picomolar potency, which is comparable to the potency of the best-in-class mAbs. All the 11 mAbs retained potent activity against the Alpha variant, while 7 also neutralized the Delta variant in the picomolar range. In contrast, most of the mAbs were ineffective against the Beta variant with only four retaining high neutralizing activity. Of note, one of them, NA8, neutralized Beta at picomolar concentration. Impressively, NA8 also potently neutralized both Omicron BA.1 and BA.2 sublineages with a picomolar potency, as well as the recently emerged BA.2.12.1 and BA.4 sublineages at nanomolar concentration. Since clinically approved mAbs are generally used in combination to ensure coverage against multiple variants, we compared side-by-side the neutralization potency of the combination of NA8 and NE12, used at equimolar concentrations (1:1), with that of 6 clinically approved antibodies used in paired combinations. Only the combination of NA8 with NE12 retained high neutralization potency against Omicron BA.1. Against the BA.2 sublineage, the NA8-NE12 combination was again the most potent, but a strong neutralization was also observed with the COV2-2130/COV2-2196 combination. Against the most recent variants, BA.2.12.1 and BA.4, the NA8/NE12 combination retained neutralizing activity at low nanomolar concentrations as did the REGN-10933/REGN-10987 combination, while COV2-2130/COV2-2196 was the most potent combination and LY-CoV555/LY-CoV016 was ineffective. These results suggest that NA8 and NE12, when used in combination, are one of the most broad and potent antibody pairs with picomolar or low nanomolar neutralizing activity against the major SARS-CoV-2 variants of concern. Based on these results, NA8 and NE12 were selected for further characterization and preclinical testing. C. Structural analysis of antibodies NE12 and NA8. To gain structural insight into the interactions of antibodies NE12 and NA8 with the SARS-CoV-2 spike glycoprotein, in collaboration with Drs. Kwong and Tsybovsky from the VRC, we obtained cryo-EM reconstructions of Fab NE12 and Fab NA8 bound to a stabilized spike trimer (S-6P) at nominal resolutions of 3.1 and 2.9 , respectively. This analysis showed that the structural basis for the breadth of neutralization of NA8, which has mixed features of both Barnes class-2 and class-3 antibodies, lies in the high conservation of its binding epitope, as revealed by cryo-EM analysis. The NA8 epitope is distinct from those of other mAbs hitherto reported in that, despite including a portion of the receptor-binding ridge, it is shifted toward the outer side of the RBD, allowing the antibody to sidestep the residues that are mutated in the Beta and Omicron variants. The second mAb, NE12, has features of a Barnes class-2 antibody and binds to an epitope that largely overlaps with the ACE2 receptor footprint on the RBD. 5. Antibodies NE12 and NA8 protect hamsters from SARS-CoV-2 infection. In collaboration with Dr. Buchholz, we tested the in vivo prophylactic efficacy of mAbs NE12 and NA8 in the golden Syrian hamster model, which closely mimics the severity of the disease in humans. Treatment with NA8 significantly protected hamsters from weight loss induced by two viral variants, WA-1 and Beta, while NE12 was highly effective against the WA-1 strain. Accordingly, the viral titers were significantly reduced in both the lungs and the nasal turbinates. Overall, these data demonstrate that mAb NA8 exerted strong prophylactic protection in vivo from two antigenically distinct SARS-CoV-2 variants, and NE12 exerted strong prophylactic protection against the original WA-1 strain in a highly susceptible animal model, despite the relatively low antibody doses used. 6. Antibodies NE12 and NA8 have therapeutic activity against SARS-CoV-2 in hamsters. The therapeutic efficacy of NE12 or NA8 was evaluated by administering the antibodies via the IP route 24 hours after intranasal instillation, which is a very stringent model given the extremely rapid kinetics of replication of SARS-CoV-2. NA8 significantly reduced the weight loss caused by infection with the Beta variant, while no effect of either NE12 and NA8 was seen on weight loss induced by the WA-1 strain. Analysis of viral titers in nasal turbinate and lung tissues showed a significant reduction in both NE12- and NA8-treated animals infected with WA-1 and the Beta variant in both nasal turbinate and lung tissues. Altogether, these results demonstrate that both NE12 and NA8 exerted strong therapeutic effects against sensitive SARS-CoV-2 strains in a suitable preclinical model.