Increased Antibody Avidity and Cross-Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2 Variants by Hyperimmunized Transchromosomic Bovine–Derived Human Immunoglobulins for Treatment of Coronavirus Disease 2019

Abstract Passive antibody immunotherapeutics directed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are promising countermeasures for protection and treatment of coronavirus disease 2019 (COVID-19). SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) can impact the clinical efficacy of immunotherapeutics. A fully human polyclonal antibody immunotherapeutic purified from plasma of transchromosomic (Tc) bovines hyperimmunized with SARS-CoV-2 WA-1 spike (SAB-185) is being assessed for efficacy in a phase 2/3 clinical trial when different circulating SARS-CoV-2 variants predominated. We evaluated antibody binding, avidity maturation, and SARS-CoV-2 VOCs/VOIs virus-neutralizing capacity of convalescent plasma compared with different lots of SAB-185 and individual Tc bovine sera sequentially obtained after each vaccination against Alpha, Epsilon, Iota, Gamma, Beta, Kappa, and Delta variants. In contrast to convalescent plasma, sera and SAB-185 derived from hyperimmunized Tc bovines demonstrated higher antibody avidity and more potent cross-neutralizing activity of VOCs/VOIs. Thus, SAB-185 is a potential promising therapeutic candidate for the treatment of patients infected with SARS-CoV-2 variants.

Emergency Use Authorized or phase 2/3 clinical stage passive immunotherapeutic antibody products that have been or are used to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include polyclonal antibody (pAb) derived from convalescent plasma (CP) from individual donors or immune intravenous immunoglobulin (IVIG) produced from pooled lots of large numbers of immune plasma from convalescent donors [1,2], and monoclonal-based therapeutics that target 1 or 2 epitopes on the SARS-CoV-2 spike protein. Recently, mutant (or variant) SARS-CoV-2 strains with increased infectivity and virulence that evade endogenous and/or exogenous polyclonal or monoclonal antibodies have emerged [3,4]. These SARS-CoV-2 variants have been classified by the World Health Organization as variants of concern (VOCs) or variants of interest (VOIs), with the Delta variant becoming dominant nationally and globally (https://www.who.int/en/activities/ tracking-SARS-CoV-2-variants/) [5,6].
With the emergence of SARS-CoV-2 variants and continuous waves of pandemic cluster outbreaks globally, therapeutic antibody products with high neutralizing titer and the capacity to neutralize emerging SARS-CoV-2 variants are critically required for effective treatment of coronavirus disease 2019 . It was observed that SARS-CoV-2 vaccination elicited broad cross-neutralization activity against emerging SARS-CoV-2 variants compared with convalescent COVID-19 sera [7,8]. Therefore, we hypothesized that hyperimmunization could induce high-avidity cross-neutralizing human antibodies that could be harnessed for manufacturing of hyperimmunoglobulins with broader coverage of emerging SARS-CoV-2 variant strains.
A hyperimmune anti-SARS-CoV-2 pAb product (SAB-185) is in late-stage clinical development. This product was produced from a transchromosomic (Tc) bovine platform that can produce anti-target human polyclonal immunoglobulin Gs (IgGs) in large quantities after hyperimmunization with the target antigen(s) [9,10]. Tc bovines have been generated in which both bovine immunoglobulin heavy chain loci and the lambda light chain locus were homozygously inactivated by gene targeting. Previously, human pAb IgGs against Middle East respiratory syndrome coronavirus (MERS-CoV) and Mycoplasma hominis were generated in Tc bovines, and both pAb products were shown to be safe and nonimmunogenic in phase 1 or 1b clinical trials [9,11]. SAB applied this technology to develop a therapeutic treatment for COVID-19, termed SAB-185 [8][9][10], which has been evaluated in phase 1 and 2 clinical trials and is under evaluation in a phase 3 clinical trial (ClinicalTrials.gov identifiers NCT04468958 and NCT04469179).
To produce SAB-185, the sequence encoding the extracellular domain of the Wuhan-strain spike protein was cloned into a plasmid DNA (pDNA) construct, and recombinant protein was produced using an ExpiSf9 expression system. After hyperimmunization against the spike protein using a prime (pDNA)-boost (protein) strategy, SAB-185 was purified from pooled Tc bovine plasma. While the SARS-CoV-2 VOCs and VOIs were found to have decreased susceptibility to 1 or more types of postinfection human plasma-generated antibody treatments [12][13][14][15][16][17][18], the impact of such variants on SAB-185 required investigation.
In this study, we determined the neutralization capacity of the Tc bovine sera and different lots of SAB-185 against the immunization-homologous SARS-CoV-2 WA-1 strain and several VOCs/VOIs (B. ) in a pseudovirion neutralization assay (PsVNA). In addition, we analyzed antibody binding titer and antibody avidity maturation of the Tc bovine sera and different lots of SAB-185 against prefusion spike trimers derived from the WA-1, Alpha, and Beta strains by surface plasmon resonance (SPR)-based antibody kinetics assay. As comparator, CP samples from recovered COVID-19 patients were evaluated, as endogenous anti-SARS-CoV-2 antibody avidity to prefusion spike has been reported to provide clinical benefit to human against COVID-19 [19][20][21][22].

Study Design
Tc bovine sera tested in the study were generated by immunization of Tc bovines with 2 doses of plasmid DNA encoding spike protein of SARS-CoV-2 WA-1 strain at a 3-week interval, followed by 5 doses of WA-1 recombinant spike protein at 4-week intervals. Sera from 2 hyperimmunized bovines were collected after each immunization (V1-V7). Three samples of purified human immunoglobulin were produced from plasma collected at postimmunization V3-V5. SAB-185 (V3) and SAB-185 (V4) were purified from pooled V3 plasma and pooled V4 plasma, respectively. SAB-185 (V3-V5) was purified from pooled V3, V4, and V5 plasma. For purification, pooled Tc bovine plasma was fractionated by caprylic acid. The fractionated sample containing Tc bovine-derived human IgG was further purified by affinity chromatography, first using an anti-human IgG kappa light chain-specific column to remove bovine plasma proteins, and second, by passing through an anti-bovine IgG heavy chain-specific affinity column to further remove residual bovine IgG or chimeric IgG (containing bovine heavy chain and human kappa light chain) molecules. The animal care and use protocol meets National Institutes of Health (NIH) guidelines and complied with all ethical regulations.
Seven CP lots were obtained from recovered hospitalized COVID-19 patients between May and July 2020, prior to emergence of Alpha or other VOCs. The CP was collected from unselected plasma donors in the United States (US) at least 30 days after recovery of disease.

Ethics Statement
This study was approved by the US Food and Drug Administration's Research Involving Human Subjects Committee (RIHSC #2020-04-02). Samples were collected from convalescent patients who provided informed consent to participate in the study. All assays performed fell within the permissible usages in the original informed consent.

Lentivirus Pseudovirion Neutralization Assay
Pseudovirion production and PsVNA was described in previous publications from our laboratory against SARS-CoV-2 WA-1 and variants, including B.  [19,21,23]. Pseudovirion used in the study were bearing the SARS-CoV-2 spike from the WA-1 strain and with spike mutations shown in Supplementary Table 1. The PsVNA using 293-ACE2-TMPRSS2 cell line was described previously [4,19,23,24]. All sera were heat-inactivated prior to use in the assay.
The limit of detection for the neutralization assay is 1:10. Neutralizing antibody titers (PsVNA 50 ) were measured as sample dilution that resulted in 50% reduction in viral titers. A PsVNA 50 titer >1:60 was used as a seropositive cutoff based on current understanding of neutralizing antibody as correlate of protection against COVID-19 [25].

Proteins
The SARS-CoV-2 spike plasmid encodes residues 1-1208 of the genetically stabilized prefusion 2019-nCoV_S-2P spike ectodomain fused to 8xHisTag, which was a kind gift from Barney Graham (earlier at Vaccine Research Center, NIH) [26]. This expression vector was used to transiently transfect FreeStyle293F cells (ThermoFisher; catalog number R79007) using polyethylenimine (Polysciences Inc). Protein was purified from filtered cell supernatants using StrepTactin resin (Cytiva, catalog number 29401326) and subjected to additional purification by size-exclusion chromatography in phosphate-buffered saline (PBS). HEK-293 mammalian cells producing SARS-CoV-2 prefusion spike of B.1.1.7 and B.1.351 were purchased from Acros Biologicals Inc. The native receptor-binding activity of the spike proteins was determined by binding to the human ACE2 protein.

Antibody-Binding Kinetics to SARS-CoV-2 Spike by SPR
SARS-CoV-2 antibody-binding kinetics by SPR was described in previous publications from our laboratory [19,21,23,27]. In brief, serially diluted 5, 1, or 0.2 mg/mL of Tc bovine sera or purified SAB-185 or a 10-, 50-, or 250-fold dilution of CP in BSA-PBST buffer (PBS pH 7.4 buffer with Tween-20 and bovine serum albumin [BSA]) was injected at a flow rate of 50 µL/minute (120 seconds' contact duration) for association, and disassociation was performed over a 600-second interval using a ProteOn SPR (Bio-Rad). Responses from the SARS-CoV-2 protein surface were corrected for the response from a mock surface and for responses from a buffer-only injection. The protein density on the chip was optimized to measure monovalent interactions independent of the antibody isotype. Total antibody binding was calculated with BioRad ProteOn manager software (version 3.1). All SPR experiments were performed twice, and the researchers performing the assay were blinded to sample identity.
Antibody off-rate constants, which describe the stability of the antigen-antibody complex (ie, the fraction of complexes that decays per second in the dissociation phase), were determined directly from the human polyclonal sample interaction with recombinant purified SARS-CoV-2 proteins using SPR, only for the sensorgrams with Max resonance units (RU) in the range of 5-150 RU and calculated using the BioRad ProteOn manager software for the heterogeneous sample model as previously described [19,21,23,[27][28][29]. Off-rate constants were determined from 2 independent SPR runs. Previously we confirmed that antigenantibody binding off-rate of purified IgG from plasma and fragment antigen-binding (Fab) fragments with prefusion spike under optimized conditions in SPR were very similar when adjusted for molecular weight of the bound IgG and Fab molecules. The variation for each sample in duplicate SPR runs was <5%. The data shown are the average value of 2 experimental runs.
For comparison, neutralization assays were performed on 7 human CP samples obtained at least 30 days postdischarge from recovered hospitalized COVID-19 patients between May and July 2020, prior to the emergence of VOCs against the panel of pseudovirion SARS-CoV-2 strains (WA-1 and all SARS-CoV-2 variants). Variable PsVNA 50 titers (1:120-1:1872) were observed for the 7 CP samples against the WA-1 strain with weak to moderate neutralization ( Figure 1A and Table 1). CP samples against SARS-CoV-2 VOCs/VOIs demonstrated moderate reduction of PsVNA 50 titers against Alpha, Epsilon, and Delta variants. However, the majority of the CP samples (5/7) lost their neutralization activity against Gamma, Iota, Kappa, and Beta variants. Additionally, all 7 CP samples were seronegative against the Beta variant. Samples CP-2 and CP-5 did not neutralize any of the VOCs/VOIs, and sample CP-6 only neutralized the Delta variant ( Figure 1A and Table 1). Tc bovine sera collected at V3 surpassed the neutralization activity of CP against WA-1 and SARS-CoV-2 variants (Table 1). Compared with Tc bovine serum, the CP lots exhibited greater loss (up to 100-fold) of neutralizing activities against the SARS-CoV-2 variants ( Figure 1A and Table 1).  ) of 1 mg/mL Tc bovine sera (V1-V7) and 1:10 dilution of CP lots to purified prefusion spike of WA-1 spike and Alpha and Beta variants of concern (VOCs) by surface plasmon resonance (SPR). C, Antibody avidity (antibody-antigen dissociation rate) of each of Tc bovine serum sample to purified prefusion spike of WA-1 spike and Alpha and Beta VOCs by SPR only for the Tc bovine samples with Max RU in the range of >5 RU as described in the Methods. Numbers above the group show the mean value for each group. Statistical comparisons between Tc bovine titers vs CP titers were performed using R software. Differences were considered statistically significant with a 95% confidence interval when the P value was < .05. * P ≤ .05, * * P ≤ .01, * * * P ≤ .001, * * * * P ≤ .0001.      SARS-CoV-2 neutralizing antibodies target several sites within the spike protein including N-terminal domain, receptorbinding domain, and S2 domain [30]. Key mutations in SARS-CoV-2 VOC/VOI spike protein may lead to decreased neutralization by antibodies following infection or escape from vaccination-generated antibodies [12,31,32]. These mutations have been shown to promote neutralization escapes of postinfection, postvaccination sera, monoclonal antibodies, and CP from COVID-19 patients [33,34]. To assess the impact of key spike protein mutations on vaccination-induced antibodies in Tc bovine sera and postinfection antibodies in the CPs, we evaluated antibody binding to stabilized prefusion spike ectodomain utilizing SPR ( Figure 1B). For this analysis, we chose the Alpha VOC prefusion spike as it demonstrates similar neutralization results as with the WA-1 strain. Also, the Beta variant prefusion spike protein was selected because it is the most resistant to postvaccination/infection antibodies [32]. Consistent with PsVNA 50 results, SPR analysis demonstrated low antibody binding (<24 RU) for the V2 Tc bovine sera samples to the WA-1, Alpha, and Beta prefusion spike proteins ( Figure 1B). Longitudinally, antigen-antibody complex formation rapidly increased from V3 onward for all Tc bovine sera against the spike protein of WA-1 and variants reaching peak antibody binding at V6 ( Figure 1B). The total antibody binding of V5-V7 Tc bovine sera was significantly higher than the antibody binding of the 7 CP lots against WA-1 spike as well as both spike variants. For Tc bovine sera, antibody binding to the Alpha spike protein had minimal to no loss in activity compared with WA-1 spike; however, binding of CP antibodies decreased 1.7-fold. Consistently, binding antibodies to the Beta prefusion spike reduced approximately 2-fold for Tc bovine sera and >10-fold for the 7 CP samples ( Figure 1B). An SPR-based real-time kinetic assay can be used to determine the dissociation kinetics of antibody bound to the SARS-CoV-2 prefusion spike as a surrogate of antibody avidity. Slower antibody-antigen dissociation rates (off-rates) represent higher avidity antibodies. Antibody affinity maturation is a key aspect of an effective immune response following vaccination or infection, as higher antibody avidity contributes to virus neutralization ability and protection from COVID-19 [19,21]. In addition to measuring binding antibodies against the prefusion spike of WA-1, Alpha, and Beta, we measured the antibody avidity maturation of longitudinal Tc bovine plasma to these prefusion spike proteins. Previously, we showed that the antibody kinetics measured under optimized SPR conditions of monovalent interactions between antibody-antigen complex were in the SPR binding range of 5-150 RU. This range was similar to the antigen-antibody binding off-rates of the IgG and Fab interaction with protein antigens [19,21,23,29]. The avidity of Tc bovine sera to WA-1 spike increased with each immunization and reached plateau at approximately V5, indicating the impact of repeated Tc bovine immunization on antibody avidity maturation ( Figure 1C). The Tc bovine sera binding avidity to the Alpha spike was comparable with the spike of WA-1 and followed the same trend. However, the antibody avidity of the V6-V7 Tc bovine sera to the Beta prefusion spike was 4-to 7-fold lower than with the corresponding WA-1 spike protein, but significantly higher than antibody avidity of CP against the Beta spike ( Figure 1C).

Spike Derived From SARS-CoV-2 WA-1, Alpha, and Beta Strains
To assess the antibody quality in the purified SAB-185 cGMP lots, the total antibody binding and avidity to prefusion spike of WA-1, Alpha, and Beta variants were examined with SPR analysis. The hyperimmunized SAB-185 (V4) and SAB-185 (V3-V5), which had high neutralization titers, exhibited strong antibody binding and high avidity to all prefusion spike proteins ( Figure 2B and 2C). SAB-185 (V3) showed approximately 2.2-fold lower antibody binding ( Figure 2B) and 9.6-fold lower antibody avidity (0.00496 per second) against WA-1 prefusion spike than did the other 2 hyperimmunized SAB-185 IgG (0.00053 and 0.00036 per second, respectively) ( Figure 2C). All 3 SAB-185 IgG samples showed minimum to no decrease of antibody binding to the Alpha spike ( Figure 2B) and <2-fold decrease in antibody avidity (increased dissociation rate) to the Alpha spike compared with the WA-1 spike protein ( Figure 2C). Antibody binding of all 3 SAB-185 lots to the Beta prefusion spike decreased by approximately 1.8-fold compared with the WA-1 spike. In addition, the antibody avidity to the Beta prefusion spike decreased by 3-to 4-fold compared with the WA-1 spike.

DISCUSSION
With the continued spread of SARS-CoV-2 variants worldwide, an effective therapeutic treatment against SARS-CoV-2 VOCs/ VOIs is urgently needed. It is critical to develop and evaluate broadly neutralizing antibody therapeutics against both circulating SARS-CoV-2 strains and the VOCs/VOIs. Tc bovines have been used to produce high-titer and/or high-avidity polyclonal human antibodies against viral targets such as MERS-CoV and Ebola [10,[35][36][37]. Historically, multiple polyclonal immunoglobulin products have been produced in animals and humans. Animal-derived immunoglobulins have the advantage of being a high-volume source, but the disadvantage of known ability to cause severe hypersensitivity reactions as their heterologous antibodies are highly immunogenic in humans. Humanderived immunoglobulins are generally nonimmunogenic in human recipients but have the limitation of needing to acquire a large number of donors (and plasma) with a sufficient titer against the target of interest. The use of Tc bovines to produce specific human immunoglobulins has the potential of combining the benefits of animals and humans. Prime-boost vaccination strategies with alternate vaccine platforms or different variant antigens and/or adjuvant combinations could be used in this Tc bovine model to potentially generate higher titers of broadly neutralizing human antibodies.
In this study, we assessed the longitudinal antibody responses in sera from Tc bovine animals that were hyperimmunized with the WA-1 spike. The Tc bovine serum was compared with CP samples from recovered COVID-19 patients utilizing assays that measure antibody binding and neutralizing capacity against the vaccine-homologous WA-1 strain and the SARS-CoV-2 VOCs/VOIs. The reduction in Tc bovine serum neutralization against VOCs/VOIs are in line with data from vaccinated human sera following repeated immunizations with SARS-CoV-2 mRNA vaccines (mRNA-1273 [Moderna] or BNT162b2 [Pfizer]) that demonstrated a similar decrease in fold reduction against variants [32]. The antibody-binding, neutralization titers, and antibody avidity all increased against both the SARS-CoV-2 WA-1 strain and the variant strains with each sequential WA-1 spike protein immunization. Sera derived from Tc bovine hyperimmunization exhibits advantages over postinfection antibody products, such as human CP or IVIG products, which often show low antibody avidity and  7 and B.1.351 variants of concern (VOCs). C, Ab avidity (Ab-antigen dissociation rate per second) of each of SAB-185 sample to purified prefusion spike of WA-1 spike and Alpha and Beta VOCs by surface plasmon resonance as described in the Methods.
In summary, compared with postinfection human CP lots, both hyperimmunized Tc bovine sera and purified SAB-185 demonstrated high antibody avidity and high neutralizing capacity against emerging VOCs/VOIs: Alpha, Epsilon, Iota, Gamma, Beta, Kappa, and Delta strains. Thus, anti-SARS-CoV-2 purified SAB-185 may likely lead to effective virus neutralization and protection against emerging SARS-CoV-2 strains and could potentially serve as an effective therapy for treatment of patients with COVID-19, even those infected with circulating SARS-CoV-2 VOCs/VOIs.