Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium

Summary The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31–36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%–50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.


Correspondence erica@lji.org
In brief Callaway et al. examine a panel of 397 SARS-CoV-2 antibodies to understand how some antibodies, generated early during the COVID-19 pandemic, maintain the ability to neutralize Omicron variants despite extensive mutation in their epitopes. Antibodies against highly mutated epitopes retain neutralization by binding bivalently: cleavage to Fab eliminates neutralization.

INTRODUCTION
Since emerging in late 2019, SARS-CoV-2, the causative agent of COVID-19, has infected over 550 million people worldwide. 1 In the United States alone, over 1 million people have died from COVID-19, and over 6 million people have died worldwide. 1,2 As of July 2022, 10 vaccines received WHO emergency use authorization (EUA), and more than 12 billion doses have been delivered. 3 However, a large proportion of the world's population remains unvaccinated. Current vaccines are substantially less effective against Omicron and emerging SARS-CoV-2 variants, although bivalent formulations that recently received EUA may provide better protection. 4 Vaccines still reduce disease severity, but with continued high infection rates, individuals who are immunocompromised, who cannot be vaccinated, or who have other risk factors associated with severe disease will need access to prophylactic and therapeutic interventions that retain efficacy. Monoclonal antibody (mAb) therapeutics against the SARS-CoV-2 spike protein are a key prophylactic or therapeutic option for at-risk patients and for vaccine breakthrough. During the course of infection, antibodies mechanically neutralize virions by blocking receptor binding, crosslinking viral proteins, preventing fusion with the host cell membrane, or targeting virions and infected cells for immune clearance. However, the multiple mutations in the SARS-CoV-2 spike protein of the Omicron variant have rendered most existing mAb therapeutics ineffective.
Omicron BA.1 (also known as B.1.1.529) was first reported to the World Health Organization in late November 2021 and was declared a variant of concern (VoC) days later. 5,6 Omicron's high transmission rate (R 0 > 3) and rapid doubling time contributed to its becoming the dominant VoC within about 5 weeks of its discovery. 2,3 With 62 non-synonymous mutations (36 in spike), Omicron has substantially more mutations than Alpha (24 mutations, 10 in spike), Beta (20 and 10), Gamma (24 and 10), or Delta VoCs (22 and 9) (Figure 1). Of the 36 mutations in the Omicron spike protein, 10 are in the N-terminal domain (NTD), and 11 are in the S2 subunit, which controls fusion. The remaining 15 mutations are all in the receptor-binding domain (RBD), which interacts with the angiotensin-converting enzyme 2 (ACE2) receptor. These RBD mutations include G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H ( Figure 1). Despite these substitutions, the Omicron spike still binds ACE2 with high affinity. 7,8 The BA.1.1 sublineage emerged largely in parallel with BA.1, with an array of essentially identical mutations, but carries an R346K mutation and lacks the K417N mutation in the RBD. BA.1 and BA1.1 co-dominated until mid-February 2022, when another sublineage, BA.2, began to surge. BA.2 has several new mutations in both the RBD and NTD (including T367A, D405N, and R408S in the RBD) but lacks the G446S and G496S mutations of BA.1. and BA.1.1. The sublineages BA.4 and BA.5, which contain L452R and F486V mutations in the RBD but lack Q493R and G496S, are now the dominant source of new infections for which virus sequence information is available. 9 BA.2.12.1, another Omicron lineage, has a spike sequence similar to that of BA.4 and BA.5. but has Q493R and L452Q RBD mutations and lacks the F486V mutation.
In addition to rendering most existing antibody therapeutics ineffective, Omicron spike mutations also challenged host antibody responses. Sera from individuals who received two doses of mRNA-based vaccines were 25-fold less potent against Omicron, [10][11][12][13] and most vaccinated individuals remain susceptible to Omicron infection. 14 Because SARS-CoV-2 likely will continue to accumulate mutations as it adapts to human hosts and undergoes selective pressure, vaccines and antibody therapies that are mutation resistant are urgently needed. Achieving durability requires better understanding of how some antibodies maintain neutralization capacity in the face of numerous point mutations in their epitopes.
To identify Omicron-neutralizing antibodies, we tested 397 samples contributed to the Coronavirus Immunotherapeutic Consortium (CoVIC) panel for neutralization against pseudoviruses displaying spike from Omicron and its sublineages. The CoVIC is an international effort designed to broadly characterize the antibody landscape against SARS-CoV-2 and compare candidate antibody therapeutics side by side. 15 The CoVIC panel comprises contributions from 60 different companies or academic laboratories across four continents. Included in the panel are natural monoclonal immunoglobulin Gs (IgGs), bispecific antibodies, nanobodies, polyclonal products, and engineered multivalent binding constructs. Multiple approaches were used to generate the candidate therapeutics in the panel, which originated from survivors of COVID-19, survivors of SARS-CoV-1 in 2003, or immunized wild-type and humanized mice or were engineered using in silico methods.
Antibodies in the CoVIC were analyzed in a high-throughput, high-resolution competition analysis using surface plasmon resonance to sort the panel into competition groups, particularly those antibodies that target the RBD. These analyses identified seven major epitope communities among RBD-reactive antibodies, termed RBD-1-7 (Figures 1 and S1). The seven communities and their subgroups provide finer divisions than previous characterizations and identify important functional differences. (C) Binding footprints are outlined in colors corresponding to the RBD antigenic site and were approximated by docking a Fab into a negative-stain electron microscopy map of spike bound to a representative antibody from the indicated epitope community. *The BA1.1 clones used in this study contain the K417N mutation in the RBD, but the consensus sequence for all BA1.1 isolates does not. See also Figure S1. RBD-1 overlaps the receptor-binding motif (RBM) almost exactly, whereas RBD-2 antibodies target an epitope that is shifted slightly toward the outer face relative to RBD-1. Antibodies in the RBD-3 community have footprints shifted toward N501. Together, RBD-1-3 correspond to functional subdivisions of the ''class 1'' antibodies described by Barnes et al. 16 RBD-4 and -5 antibodies both bind the outer face of RBD and are similar to class 2 and class 3, respectively. Meanwhile, RBD-6 and -7 are related to class 4 but are functional subdivisions of that class, according to their competition profile.
We previously described RBD epitope communities for 176 CoVIC antibodies 15 and have since assigned 195 more antibodies to epitope communities, which resulted in a more finely detailed classification scheme. Now, all epitope communities except for RBD-1 and -3 have several subclusters that were defined based on their competition with other antibody communities. In particular, RBD-4 and -5 epitope communities now have additional subdivisions, and RBD-2b.1, -2b.2, and -2b.3 communities have been renamed as RBD-2c, -2d, and -2b, respectively, to better highlight their distinct epitope subgroups and relationship to other communities.
To determine what types of antibodies in the therapeutic landscape retain neutralization activity against Omicron subvariants and understand how epitope community is related to potency against Omicron, we tested the CoVIC antibodies in a neutralization assay using pseudovirus bearing Omicron spikes from BA. 1 Article ll OPEN ACCESS determine neutralization titer. We then examined how retention of Omicron neutralization relates to competition group and antibody structure, as well as other antibody features. The results of this study provide information that can be used to guide both therapeutic selection and vaccine design.

RESULTS
We first tested 397 antibodies in the CoVIC panel series in a block neutralization assay. Antibodies were aliquoted into 96-well plates at two concentrations (25 mg/mL and 250 ng/ mL) and then incubated with vesicular stomatitis virus (VSV)based pseudovirus displaying spike protein from either D614G, the Beta (B.1.529), Delta, or Mu VoCs, or one of three Omicron lineages: BA.1 (also known as B.1.1.519), BA1.1, or BA.2. This initial analysis allowed rapid identification of antibodies that retained detectable potency against Omicron.
At 250 ng/mL, 66 antibodies in the panel ($16%) had potent neutralization against BA.1, resulting in %20% of infected cells relative to cells infected in the absence of antibody (Figures 2 and S2; Table S1). Another 14 ($3%) were less potent but still had detectable activity, with 20%-40% of cells infected. Of the 66 antibodies that potently neutralize BA.1, six belong to RBD-1, 17 to RBD-2, one to RBD-3, eight to RBD-4, one to RBD-5, 31 to RBD-7, and one to a trimer-specific site. The remaining antibody did not have a binding site assigned. The 14 antibodies with intermediate potency included one RBD-1, four RBD-2, three RBD-4, two RBD-5, two RBD-7, one S2 binder, and one unassigned antibody.

Mutations that affect antibody neutralization
The three Omicron variants in the block neutralization assay contain 15, 16, and 16 mutations in the RBD for BA.1, BA1.1, and BA.2, respectively, compared with D614G spike. The BA.1 and BA1.1 examined here differ by a single residue at 346 and have similar neutralization profiles (Figures 1 and 2). BA.2 shares many of its mutations with BA.1 but has additional mutations at residues 376, 405, and 408 and lacks mutations at residues 446 and 496 ( Figure 1). As a result, BA.2 shows a substantially different neutralization profile than BA.1 and BA1.1 ( Figure 2). Notably, antibodies in the RBD-7 community neutralize BA.1 and BA1.1 well but do not neutralize BA.2 ( Figure 2). Conversely, several RBD-4 and -5 community members neutralize BA.2 well but not BA.1 or BA1.1 ( Figure 2). The loss of BA.2 neutralization in the RBD-7 community is likely due to the T376A and/or D405N point mutations in the RBD-7 antigenic site, while some antibodies in the RBD-4 and -5 communities likely regain neutralization because BA.2 lacks G446S, G496S, and R346K mutations, which overlap the RBD-4 and -5 antigenic sites ( Figure 1).
Examining antibody neutralization profiles and RBD differences between SARS-CoV-2 variants yields additional insight into how mutations affect antibody neutralization. For example, multiple members of the RBD-2c epitope community neutralize D614G, Delta, and Mu strains well but not Beta. The only RBD mutation unique to Beta, K417N, lies within the RBD-2 epitope and likely disrupts binding by these antibodies. Similarly, we can conclude that many members of the RBD-1 and -2a communities are sensitive to mutations at 484 and 501 (and possibly 417).

BA.4/5 and BA.2.12.1 neutralization
We further tested the neutralization activity of 20 antibodies that potently neutralized D614G, Beta, Delta, Mu, BA.1, BA1.1, and BA.2 against pseudovirus bearing BA.4/5 and BA2.12.1 spike. Although neutralization titers were lower against BA2.12.1 compared with the parent D614G strain for multiple antibodies, all maintained potent neutralization against BA.2.12.1 (Figure S3). For BA.4/5, however, six antibodies lost neutralization and another eight could not completely neutralize virus at the highest antibody concentration tested ( Figure S3). This loss of activity is likely due to the F486V RBD mutation, which is unique to the BA.4 and BA.5 sublineages. The remaining seven antibodies were unaffected by the F486V mutation and neutralized BA.4/5 pseudovirus as well as or better than the D614G parent strain ( Figure S3).

Bivalent binding
Structural biology has traditionally studied the antigen-binding fragment (Fab) and considered single-Fab footprints when evaluating breadth of neutralization. Here, we evaluated intact IgG, the biologically relevant form that protects after infection, vaccination, or prophylactic or therapeutic delivery of antibodies. We examined the binding interactions between Omicron-neutralizing IgG and spike using negative-stain electron microscopy. We chose 18 antibodies that neutralize Omicron well and cover a representative range of the Omicron-neutralizing epitope groups, with two RBD-1 community members, ten RBD-2, one RBD-3, three RBD-4, one RBD-5, and one RBD-7. RBD-2 is the most abundant epitope group in the experiment and was also the most common epitope targeted by the therapeutic candidates. Although RBD-7 contains multiple antibodies that retain Omicron neutralization, most members of the RBD-7 group are not natural IgGs but instead are engineered, tetravalent assemblies of heavy chain-only nanobody (VHH) or scFv domains. When incubated with spike ectodomains, these molecules form large aggregates that are unsuitable for structural analysis. For the RBD-7 sample selected, CoVIC-63, we first digested the molecule and purified the individual VHH domains before forming complexes with spike in order to prevent formation of cross-linked aggregates. 15 We found that 13 of the 18 Omicron-neutralizing antibodies ($70%), including all of the RBD-2 binding antibodies, bind spike bivalently, with each arm of the antibody engaging a neighboring RBD in spike ( Figure 3A). All of the RBD-2 bivalent antibodies bound two RBDs in the ''up'' conformation, while the RBD-3, -4, and -5 bivalent antibodies each bound two RBDs in the ''down'' conformation. Of the remaining five non-bivalent, Omicron-neutralizing antibodies, two are ACE-2-Fc constructs and one is a multivalent RBD-7, leaving the two RBD-4b binders as the only natural antibodies that bind monovalently and neutralize Omicron. These RBD-4b binders likely maintain Omicron neutralization despite their monovalent interaction because the RBD-4 footprint has greater sequence conservation between D614G and the Omicron variants. In the RBD-4 footprint, there are only three Omicron substitutions: R346K, G446S, and Q498R. Each is either a biochemically conserved mutation (346 and 446) or at the edge of the binding footprint (498) (Figure 1).
Our previous electron microscopy work with spike-binding antibodies in CoVIC showed that antibodies from different epitope communities approach the RBD from different angles. The approach angle and resulting position of the rest of the IgG determine whether the antibody binds to spike monovalently, bivalently within one spike (intra-spike crosslinking), or by connecting two different spikes (inter-spike crosslinking). 15 In particular, many RBD-2 and some RBD-5 community members tend to bind bivalently, with intra-spike crosslinking. The remaining epitope communities typically bind monovalently (one Fab to one spike, with the other Fab free) or crosslink separate spike trimers (each Fab binds a different spike).
We hypothesized that bivalent binding, which would increase the strength of interaction between an antibody and a spike protein, may allow these antibodies to retain neutralization activity toward Omicron despite its many mutations. To test this hypothesis, we first used negative-stain electron microscopy to determine the extent to which bivalency is linked to Omicron neutralization. In this analysis, we selected an additional nine antibodies from the RBD-2 community that failed to neutralize Omicron. For these antibodies, eight of the nine bind entirely or partially monovalently, with 22%-100% of the antibody/spike complexes comprising monovalently bound spikes ( Figure 3B). In contrast, each RBD-2 antibody that does neutralize Omicron exhibits only bivalent binding, with no monovalent binding interactions visible in any 2D class ( Figure 3A).

Impact of bivalency on neutralization
To further test the importance of bivalent binding for Omicron neutralization, we isolated Fab fragments from a subset of the Omicron-neutralizing antibodies and compared the D614G and BA.1 pseudovirus neutralization activity of Fabs with that of the corresponding intact IgG. In this analysis, we included six RBD-2 bivalent binders, as well as one RBD-3 bivalent binder and one RBD-4 monovalent binder as controls. All eight IgGs neutralize D614G pseudovirus well (Figure 4). Five of the eight IgGs neutralize BA.1 and D614G equally well, while the remain-ing three (CoVIC-292, -334, and -260) have a 6-to 24-fold reduction in neutralization activity for BA.1 pseudovirus compared with D614G ( Figure 4).
Against D614G pseudovirus, all Fabs have lower neutralization activity compared with intact IgG (Figure 4), suggesting, as expected, that inter/intra-spike crosslinking or steric hindrance imposed by the larger mass of the IgG molecule can inhibit viral infection. However, digestion of the bivalently binding IgGs into Fabs disproportionately decreases neutralization of BA.1 pseudovirus compared with the monovalent binding control CoVIC-295 ( Figure 4). While CoVIC-295 Fabs neutralize D614G and BA.1 pseudovirus equally (albeit to lower levels than CoVIC-295 IgGs) (Figure 4), Fabs from bivalent-binding antibodies all neutralize BA.1 pseudovirus worse than D614G pseudovirus, and some fail to neutralize BA.1 pseudovirus at all. This effect is particularly pronounced for CoVIC-290, -333, and -361. For these antibodies, the IgGs neutralize both D614G and BA.1 with essentially equal IC 50 values, but the Fab fragments exhibit either a 20-fold reduction in neutralization relative to D614G or do not neutralize BA.1 at all (Figure 4).

Function of bivalent binding
To confirm that the disproportionate loss of Omicron pseudovirus neutralization is due to loss of avidity after IgGs are digested into Fabs, rather than to crosslinking or steric hindrance, we used surface plasmon resonance (SPR) to measure the binding kinetics between BA.1 spike trimers and six sets of IgGs and the corresponding Fabs. We also measured binding affinity between D614G spike and IgGs. Five sets of IgGs/Fabs (CoVIC-290, 292, -333, -334, and -368) bind bivalently to the RBD-2b or -3 epitopes. The sixth set (CoVIC-295) serves as a control and binds monovalently to RBD-4b, a footprint that has a higher degree of sequence conservation than other epitopes.
Retention of binding through avidity does translate to neutralization activity. Because Fabs from CoVIC-290, -292, and -334 bind BA.1 spike poorly ( Figure 5B), the Fabs fail to neutralize BA.1 pseudovirus (>10,000 ng/mL IC 50 ; Figure 4). Their bivalent IgGs, in contrast, which bind BA.1 spike with higher affinity, potently neutralize Omicron pseudovirus with IC 50 values of 25, 73, and 70 ng/mL for CoVIC-290, -292, and -334, respectively (Figures 4 and 5B). However, the benefits of bivalent binding go beyond increased binding affinity. When we compare CoVIC-333 and -295, which bind BA.1 spike strongly both as IgG and as Fab, we note a difference in neutralization outcome Fab fragments from both antibodies bind BA.1 with high affinity and to similar levels ($1.5-fold affinity difference) ( Figure 5B), but Fabs from the monovalent binder CoVIC-295 neutralize BA.1 and D614G pseudovirus equally (Figure 4), and Fabs from the bivalent binder CoVIC-333 neutralize BA.1 pseudovirus neutralization much more poorly than D614G (Figure 4). However, CoVIC-333 IgGs neutralize D614G and Omicron pseudoviruses equally well (Figure 4). Thus, bivalent binding not only helps low-affinity Fabs to retain binding as IgG but also appears to enhance the neutralization potency of high-affinity Fabs by crosslinking spike protomers or otherwise mechanically neutralizing virus.

DISCUSSION
Since its emergence in late 2019, SARS-CoV-2 has acquired numerous mutations throughout its genome, both to its spike protein and elsewhere, that affect its infectivity and ability to evade the host immune response. These mutations contributed to waves of variants that perpetuated the pandemic. Although the Omicron variant and its sublineages are the most recent and extreme example, prior VoCs including Beta, Delta, and Mu each introduced a number of mutations that affected affinity and neutralization. Recombination between SARS-CoV-2 strains is also possible, resulting in hybrid viruses that have combinations of previously observed mutations. SARS-CoV-2 will likely continue to mutate as it adapts to humans and other host In this work, we analyzed nearly 400 antibody therapeutic candidates to learn the determinants for durable neutralization against Omicron and its sublineages, which have the greatest divergence of all the VoCs relative to the original strains. This study focused on antibodies against the RBD, as antibodies against this site exhibit the most potent neutralization, these epitopes are immunodominant, and the majority of therapeutic candidates target this domain. Epitopes in the fusion subunit S2 are more conserved but rarely neutralize as potently.
The mutations in BA.1, BA1.1, and BA.2 spikes each sharply decrease the number of antibodies capable of neutralizing these variants at therapeutically relevant concentrations. We show that mutations in Omicron affect antibodies in every RBD epitope community, as these mutations span the binding footprints of all seven epitope communities. Despite the large number of mutations, however, over 16% of the study panel maintain durable neutralization of Omicron, Beta, Delta, and Mu VoCs.
Using electron microscopy to analyze IgG interactions with spike and SPR to measure IgG and Fab binding affinity, we found that IgGs that retain robust neutralization of Omicron and other VoCs tend to bind bivalently. These antibodies attach to spike with both Fabs simultaneously, with both spike RBDs in either the up or down conformation depending on the antibody. In contrast, most monovalently binding IgGs within the same epitope group lose binding and neutralization against Omicron. Bivalent binding compensates for mutation-decreased binding affinity of each Fab to variant spike with avidity, effectively squaring the binding capacity of an individual Fab for the spike protein. When we remove the capacity for bivalent binding by digesting antibodies into Fabs, many of these antibodies lose their ability to neutralize Omicron, even when the parent IgG molecule neutralizes Omicron potently, and both Fab and IgG neutralize D614G. In short, emergence of mutations leads to loss of antibody neutralization unless the antibody recognizes a conserved epitope or can compensate for mutations with avidity, and bivalent binding may further enhance neutralization by crosslinking spike protomers or otherwise inactivating spike.
These results suggest that bivalently binding IgGs could be prioritized as therapeutics or that bi-or multivalency could be engineered into therapeutic molecules. These results further suggest that spike-based vaccine strategies, in which RBDs are displayed in the appropriate distance and orientation to elicit bivalent IgG recognition, could lead to more durable, variantresistant antibody responses. Similarly, antigen density on nanoparticle vaccines that mimics that on the virus would facilitate elicitation of durability via intra-spike bivalent binding. RBDbased vaccines, such as on nanoparticles, would benefit from ensuring spike-like spacing. Further engineering of spike protein to make the RBD conformations homogeneous (all up or all down) may further favor the generation of antibodies that can use avidity to their advantage.

Limitations of the study
Most of the antibodies examined in this study were generated relatively early in the COVID-19 pandemic, before the emergence of Omicron and many of the other VoCs. Individuals who were infected by the later VoCs or who have received Omicron booster vaccines may have developed broadly neutralizing antibodies that can better target both the original SARS-CoV-2 strain and the VoCs. It is possible that these antibodies, if they exist, neutralize virus by targeting different antigenic sites or via different mechanisms than those described in this work. The antibodies selected for this study were also selected for the ability to neutralize virus in vitro. There may also be Article ll OPEN ACCESS antibodies that protect against Omicron or other VoCs in vivo but do not neutralize in vitro, and they could have different mechanisms of action from those described here.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

CoVIC antibodies
The CoVIC antibody panel used in this study consists of 397 antibodies contributed by numerous laboratories, both industrial and academic, from five continents. The contributors to CoVIC were asked to indicate the species of origin for their antibodies. If the antibodies originated from human subjects (most were survivors of COVID-19 infection), the contributors were required to provide documentation indicating that informed consent was obtained to allow the antibodies to be used in research studies. However, the contributors were not required to provide additional information concerning patient age, gender, or other characteristics. For fairness of the study and so that all information could be made immediately and publicly available, each antibody was assigned a number, blinding it to both experimenters and contributors. While most antibodies in the panel were isolated from individuals who had been infected by or vaccinated against SARS-CoV-2, the panel also includes bispecific, multivalent, chimeric, or otherwise engineered molecules. Several antibodies were isolated and purified as previously described. 15,[17][18][19][20][21][22] To isolate one set of antibodies, hybridoma cell lines were obtained by immunizing Balb/c mice with recombinant SARS-CoV-2 Spike protein S1 (aa1-666). Following immunization, lymphocytes were harvested from lymph nodes and immortalized by fusion with a myeloma cell. Antibodies from hybridoma cell culture supernatants were purified with Protein A or G.
The amino acid sequence of another antibody was identified from B cells of an infected individual, residues analyzed for developability liabilities, and reformatted as an IgG1 isotype as synthetic genes. A Chinese hamster ovary (CHO) stable cell pool was generated to express COVIC-355. Recombinant protein for this antibody was purified from culture supernatant using protein A and cation exchange chromatography columns, according to standard techniques.
Another set of antibodies was developed using rabbit immunization followed by humanization and affinity maturation. Rabbits were immunized with wild type RBD, and a Fab-phage library was constructed and panned against wild type spike protein trimer to isolate a highly neutralizing rabbit clone. CDRs were grafted onto a human framework to construct an initial lead candidate. A stage-based affinity maturation platform was used to further humanize the CDRs and to improve breadth of neutralization by iterative enrichment on variant spike protein trimers including wild type, Alpha, Beta, Gamma, Delta, Epsilon, and Kappa. mAbs were transiently expressed in HEK293T cells and purified by protein A chromatography.
One construct was an ACE2-Ig fusion, which was purified using a HiTrap MAbSelect SuRe Protein A column (Cytiva) that was neutralized with 1 M Tris pH 9.0. Another set of antibodies was isolated from fresh blood and frozen PBMCs obtained from previously infected donors or those with active SARS-CoV-2 infections by Bloodworks Northwest as part of the ImmuneRACE study. 23,24 Fully human antibody IgH and IgKL sequences were natively paired in high throughput using a modified version of pairSEQ 25 from isolated ASCs or memory B cells enriched for spike-specificity using flow cytometry. Abundance, isotype, and patterns of somatic hypermutation were used to select candidates for testing from the resulting set of paired sequences. Variable regions of selected antibodies were cloned into pcDNA3.4 plasmid before subcloning into an IgG1 backbone and transfection into CHO or HEK 293 cell lines for antibody production. Purity was assessed by SDS-PAGE and A280 following affinity purification.
Soluble spike ectodomains for the D614G and Omicron BA.1 strains containing the HexaPro stabilizing mutations 26 were cloned into the phCMV vector and expressed in ExpiCho cells (ThermoFisher) using the HighTiter protocol according to the manufacturer's instructions. D614G HexaPro spike protein ectodomain with C-terminal foldon, HRV3C protease, 8x His-tag, and Avi-tag was used for negative stain electron microscopy. D614G and Omicron BA.1 spike protein with C-terminal Foldon, HRV3C protease, 8x His-tag,