An essential criterion for understanding the immunogenicity potential of biotherapeutic proteins is defining the set of peptides processed and presented by human leukocyte antigen (HLA) class II molecules. The heterozygotic state of most individuals and the extreme polymorphic nature of these molecules preclude efforts to define both the precise sequences presented by various alleles and the percentage of patients that are subject to immune responses that can negate clinical benefit and/or result in adverse events. We have developed a diverse, robust, and reproducible monoallelic HLA-DRB1 system in professional antigen presenting cells capable of examining the immunogenic potential of any human IgG. Determination of the allelic restriction of adalimumab CD4 T cell epitopes for two distinct geographic populations underscores the vitality of this system as a central component of a preclinical immunogenicity risk assessment strategy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.

Kathryn Westendorf, Stefanie Žentelis, Denisa Foster, Peter Vaillancourt, Matthew Wiggin, Erica Lovett, Jörg Hendle, Anna Pustilnik, J. Michael Sauder, Lucas Kraft, Yuri Hwang, Robert W. Siegel, Jinbiao Chen, Beverly A. Heinz, Richard E. Higgs, Nicole Kalleward, Kevin Jepson, Rodrigo Goya, Maia A. Smith, David W. Collins, Davide Pellacani, Ping Xiang, Valentine de Puyraimond, Marketa Ricicova, Lindsay Devorkin, Caitlin Pritchard, Aoise O’Neill, Courtney Cohen, John Dye, Kathleen I. Huie, Catherine V. Badger, Darwyn Kobasa, Jonathan Audet, Joshua J. Freitas, Saleema Hassanali, Ina Hughes, Luis Munoz, Holly C. Palma, Bharathi Ramamurthy, Robert W. Cross, Thomas W. Geisbert, Viktoriya Borisevich, Iliana Lanz, Lisa Anderson, Payal Sipahimalani, Kizzmekia S. Corbett, Lingshu Wang, Eun Sung Yang, Yi Zhang, Wei Shi, Barney S. Graham, John R. Mascola, Tara L. Fernandez, Carl L. Hansen, Ester Falconer, Bryan E. Jones, and Bryan C. Barnhart

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19. In Brief LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. Highlights LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variants No loss of potency against currently circulating variants Binding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID database Breadth of neutralizing activity and potency supports clinical development

are desperately needed, given that some of the existing therapeutic antibodies are less effective or ineffective against certain variants and the impact of variants on vaccine efficacy is still poorly understood. activity = acute 2. a IC50 values presented are geometric mean if multiple experiments were performed and a weighted geometric mean for the Wuhan strain (n=10) 95% CI values were included for variants tested only once. IC50 for ACE2-binding inhibition of Wuhan sequence is presented as the consensus estimate as of 2021 achieved across repeat experiments. b Fold shifts are calculated comparing to the in-experiment Wuhan control. The geometric mean of the fold changes is provided if multiple experiments were performed. For ACE2-binding inhibition, no inhibition represents <50% ACE2-binding inhibition with up to 3.75 μg/mL mAb. For the soluble assay, the IgG secreted by B-cells was captured on beads using the constant region. Binding to secreted IgG immobilized onto beads was subsequently assessed using soluble fluorescently labeled SARS-CoV-2 S or SARS-CoV-2 RBD antigen. Beads or cells were flowed onto microfluidic screening devices and incubated with single antibody-secreting cells, and mAb binding to cognate antigens was detected via a fluorescently labeled anti-human IgG secondary antibody, or soluble antigen labeled with fluorophore. Positive hits were identified using machine vision and recovered using automated robotics-based protocols. preparations by infection of VeroE6 cells (ATCC CRL-1586) with serially diluted virus followed by staining with an anti-luciferase antibody (Novus Cat # NB600-307PEATT594) and analysis by fluorescence-activated cell sorting using a Becton Dickinson LSRFortessaTM X-20. Particle titers were determined by isolation of genomic RNA and quantitation by RT-qPCR (NEB # E3031) using an Applied Biosystems vii7A™ Real-Time PCR system. Relative luciferase reporter signal read-out was determined by luciferase assay (Promega Cat # E2650) of extracts from VeroE6 cells infected with serially diluted virus. Luciferase activity was measured on a PerkinElmer EnVision 2104 multilabel reader. concentration of mAb tested. experimental batch, data obtained duplicate or triplicate. potent vitro in vivo activity against SARS-CoV-2.