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Severe acute respiratory coronavirus-2 (SARS-CoV-2) is a novel viral pathogen and therefore a challenge to accurately diagnose infection. Asymptomatic cases are common and so it is difficult to accurately identify infected cases to support surveillance and case detection. Diagnostic test developers are working to meet the global demand for accurate and rapid diagnostic tests to support disease management. However, the focus of many of these has been on molecular diagnostic tests, and more recently serologic tests, for use in primarily high-income countries. Low- and middle-income countries typically have very limited access to molecular diagnostic testing due to fewer resources. Serologic testing is an inappropriate surrogate as the early stages of infection are not detected and misdiagnosis will promote continued transmission. Detection of infection via direct antigen testing may allow for earlier diagnosis provided such a method is sensitive. Leading SARS-CoV-2 biomarkers include spike protein, nucleocapsid protein, envelope protein, and membrane protein. This research focuses on antibodies to SARS-CoV-2 spike protein due to the number of monoclonal antibodies that have been developed for therapeutic research but also have potential diagnostic value. In this study, we assessed the performance of antibodies to the spike glycoprotein, acquired from both commercial and private groups in multiplexed liquid immunoassays, with concurrent testing via a half-strip lateral flow assays (LFA) to indicate antibodies with potential in LFA development. These processes allow for the selection of pairs of high-affinity antispike antibodies that are suitable for liquid immunoassays and LFA, some of which with sensitivity into the low picogram range with the liquid immunoassay formats with no cross-reactivity to other coronavirus S antigens. Discrepancies in optimal ranking were observed with the top pairs used in the liquid and LFA formats. These findings can support the development of SARS-CoV-2 LFAs and diagnostic tools.

In this study, we describe the characterization and validation of a diverse panel of fully human CD3-binding antibodies, including hundreds of human and cyno cross-reactive binders. We used two proof-of-concept TCE targets to demonstrate that this panel streamlines CD3 T-cell engager (TCE) development, enabling identification of optimal tumor cell-killing and cytokine-release profiles. CD3 TCEs have potential to be powerful cancer treatments, but the small number of available CD3-binding antibodies and limited multispecific engineering technologies have been barriers to development. Identifying TCEs that balance anti-tumor potency with potential toxicities, such as cytokine release syndrome, requires simultaneous tuning of both the CD3- and tumor-binding arms. Pairs of antibodies that achieve this balance are rare, creating a need for diverse panels of developable antibodies that can be combined and tested to identify optimal clinical candidates. To streamline TCE development, we discovered a diverse panel of CD3-binding antibodies. We screened over 5 million single cells from humanized mice and identified 585 unique CD3-specific antibody sequences. Of these, over 170 were identified as cross-reactive to human and cyno CD3 in primary screening. We then used high-throughput characterization to curate a panel of diverse and developable antibodies. We found a wide range of CD3εδ and CD3εγ binding specificities, affinities, and kinetics. Epitope binning analysis revealed multiple bins containing human and cyno cross-reactive binders, some of which are distinct from previously described cross-reactive antibodies, such as SP34-2. We assessed their biophysical properties and identified antibodies with good developability properties, including high thermal stability and low hydrophobicity, self-association, polyspecificity, and aggregation. To validate these antibodies, we used OrthoMab™ to generate proof-of-concept TCE panels with fixed tumor-binding arms. We identified CD3 x EGFR TCEs with high potency, low cytokine release, functional cross-reactivity in a cyno T cell-mediated tumor killing assay, and good pharmacokinetic properties in Tg32 mice. A second proof-of-concept CD3 x PSMA panel further validated our antibodies in bispecific formats. Together, these studies demonstrate that starting with diverse CD3-binding antibodies streamlines identification of developable TCEs with optimal potency and cytokine release. We leveraged data from our extensive characterization of CD3-binding antibodies in mono- and bispecific formats to develop a strategy for down-selection and pairing of CD3- and tumor-binding antibodies, and a high-throughput method for analysis of resulting TCEs. By categorizing antibodies based on functional properties, we are able to rapidly pinpoint optimal potential clinical candidates for specific tumor targets. Citation Format: Juntao (Matt) Mai, Kate Caldwell, Lindsay DeVorkin, Grace P. Leung, Karine Herve, Yuri Hwang, Cristina Faralla, Wei Wei, Emma Lathouwers, Valentine de Puyraimond, Lauren Clifford, Rhys S. Chappell, Stefan Hannie, Katherine J. Lam, Harveer Dhupar, Tran N. Tran, Melissa Cid, Lena M. Bolten, Tova Pinsky, Ping Xiang, Courteney Lai, Ahn Lee, Vivian Z. Li, Patrick Chan, Jasmine Chin, Steve Booth, Amy C. Lee, Stephanie Masterman, Sherie Duncan, Aaron Yamniuk, Kush Dalal, Tim M. Jacobs, Raffi Tonikian, Bryan C. Barnhart. Identifying T-cell engagers with optimal potency and cytokine-release profiles with a diverse panel of CD3-binding antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1886.

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 BriefLY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated.HighlightsLY-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) variantsNo loss of potency against currently circulating variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development