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Capturing and creating a recombinant human antibody hyperimmune for treating and preventing COVID-19

Sheila M. Keating1, Rena A. Mizrahi1, Michael A. Asensio1, Emily Benzie1, Yao Chiang1, Ashley Gras1, Jackson Leong1, Renee Leong1, Yoong Wearn Lim1, Vishal A. Manickam1, Angelica V. Medina-Cucurella1, Ariel R. Niedecken1, Jasmeen Saini1, Kacy Stadtmiller1, Ellen K. Wagner1, Dirk Büscher2, Jose Vicente Terencio2, Thomas H. Oguin3, Adam S. Adler1, David S. Johnson1

1 GigaGen Inc., South San Francisco, CA, USA

2 Grifols S.A., Sant Cugat del Vallès, Spain

3 Regional Biocontainment Laboratory, Duke University Medical Center, Durham, NC, USA

Plasma-derived hyperimmune polyclonal antibodies from recovering patients are effective passive immune therapeutics. They encompass the natural immune response that has already demonstrated protective efficacy. These drugs are used to protect individuals who are at high risk of exposure or for treatment during early infection. However, the potency and supply required to treat population-level pandemic infections can be a major constraint. We used a microfluidic method to isolate individual antibody-producing cells and molecular genomics to capture individual sequences from an entire diverse mammalian antibody repertoire. From this, a recombinant, multivalent hyperimmune drug can be quickly manufactured for industrial scale production. During the first months of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic, 50 convalescent donors who were symptomatic or had tested positive at least 2 weeks before, were recruited to provide a 60mL whole blood specimen. Approximately 1x10⁷ B cells were isolated from each sample and frozen into 2 – 5 x10⁶ cell aliquots. Antibody concentrations against SARS-CoV-2 spike protein were measured for each donor and 16 of the highest titered donors were selected for the antibody cloning pipeline. In 2-member pools, cells were thawed, combined, and run through the microfluidic droplet system. Antibody sequences from each library were captured and cloned as single-chain variable fragment and expressed in yeast for selection by flow cytometry (FACS). Although Receptor Binding Domain (RBD) and Spike proteins were initially used as bait for selecting antigen-specific sequences by FACS, RBD was selected as the primary sorting antigen for the final antibody library. Analysis of the RBD-binding sequences demonstrated that the natural antibody responses were primarily an IgG1 isotype; therefore, to preserve the natural antibody function, the RBD-specific sequences were cloned on a human IgG1-isotype backbone and stably integrated at a single site for production in Chinese hamster ovary cells. The recombinant antibody pool, GIGA-2050, was analyzed for sequence diversity, antibody titer, pseudotype and live virus neutralization, and cross-variant binding and blocking. From 16 donors, this antibody development pipeline yielded 12,500 RBD-specific antibodies, the ELISA binding titer was between 99- and 747-fold more potent than matched donor convalescent plasma (MDCP). The neutralization titer was between 44- and 1,767-fold more potent than MDCP. Pseudotype-virus and live-virus neutralization results significantly correlated (r=0.96; p<0.005). GIGA-2050 demonstrated potent binding to all SARS variants, including SARS-CoV, and no antibody dependent enhancement of infection was detected. This technology enabled creation and manufacturing of a potent recombinant hyperimmune for SARS CoV-2 in less than three months, and it was significantly more potent than MDCP (p<0.005) as measured by ELISA and in vitro neutralization. This work has demonstrated that entire antibody repertoires from convalescent donors can be captured and used to quickly create and manufacture recombinant antibodies for industrial-scale production for treating an entire population at risk of pandemic infection.