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The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omicron variant was discovered and classified as a variant of concern (VOC). Omicron shows substantially more mutations in the spike protein than any previous variant, mostly in the receptor binding domain (RBD). We analyzed the binding of the Omicron RBD to the human ACE2 receptor (hACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta or Delta RBDs variants.All RBDs were produced in insect cells. RBD binding to hACE2 was analyzed by ELISA and microscale thermophoresis (MST). Similarly, sera from 27 COVID-19 patients, 58 fully vaccinated individuals and 16 booster recipients were titrated by ELISA on the fixed RBDs from the original Wuhan strain, Beta, Delta and Omicron VOC.Surprisingly, the Omicron RBD showed a weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were significantly lower against Omicron RBD compared to the original Wuhan strain. However, a difference of 2.5 times was observed in RBD binding while in other studies the neutralization of Omicron SARS-CoV-2 was reduced by a magnitude of 10x and more. These results indicate an immune escape focused on neutralizing antibodies.The reduced binding of sera to Omicron RBD adds evidence that current vaccination protocols may be less efficient against the Omicron variant.

Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen-KLK7-the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented.

The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed a sub nM IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody was demonstrated in the Syrian hamster and in the hACE2 mice model using a silenced human IgG1 Fc part. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD was solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibtion of STE90-C11 is not blocked by many known RBD mutations including N439K, L452R, E484K or L452R+E484Q (emerging B.1.617). STE90-C11 derived human IgG1 with FcγR silenced Fc (COR-101) is currently undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.In BriefHuman antibodies were selected from convalescent COVID-19 patients using antibody phage display. The antibody STE90-C11 is neutralizing authentic SARS-CoV-2 virus in vitro and in vivo and the crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD revealed that this antibody is binding in the RBD-ACE2 interface. S1 binding of STE90-C11 and inhibition of ACE2 binding is not blocked by many known RBD mutations.

Background The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omicron variant was discovered and immediately classified as a variant of concern (VOC), since it shows substantially more mutations in the spike protein than any previous variant, especially in the receptor-binding domain (RBD). We analyzed the binding of the Omicron RBD to the human angiotensin-converting enzyme-2 receptor (ACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta, or Delta RBD variants. Methods All RBDs were produced in insect cells. RBD binding to ACE2 was analyzed by ELISA and microscale thermophoresis (MST). Similarly, sera from 27 COVID-19 patients, 81 vaccinated individuals, and 34 booster recipients were titrated by ELISA on RBDs from the original Wuhan strain, Beta, Delta, and Omicron VOCs. In addition, the neutralization efficacy of authentic SARS-CoV-2 wild type (D614G), Delta, and Omicron by sera from 2× or 3× BNT162b2-vaccinated persons was analyzed. Results Surprisingly, the Omicron RBD showed a somewhat weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were significantly lower against Omicron RBD compared to the original Wuhan strain. A 2.6× reduction in Omicron RBD binding was observed for serum of 2× BNT162b2-vaccinated persons. Neutralization of Omicron SARS-CoV-2 was completely diminished in our setup. Conclusion These results indicate an immune escape focused on neutralizing antibodies. Nevertheless, a boost vaccination increased the level of anti-RBD antibodies against Omicron, and neutralization of authentic Omicron SARS-CoV-2 was at least partially restored. This study adds evidence that current vaccination protocols may be less efficient against the Omicron variant.

The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed an IC50 of 0.56 nM in a plaque-based live SARS-CoV-2 neutralization assay. The crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD was solved at 2.0 A resolution showing that the antibody binds at the same region as ACE2 to RBD. In contrast to other published anti-SARS-CoV-2 antibodies, the binding of STE90-C11 is not blocked by known RBD mutations, endowing our antibody with higher intrinsic resistance to those possible escape mutants. Funding: We kindly acknowledge the financial support of MWK Niedersachsen (14-76103-184CORONA-2/20) for antibody generation. We also acknowledge support of the European Union for the ATAC (“antibody therapy against corona”, Horizon 2020 number 101003650) consortium. Conflict of Interest: The authors declare a conflict of interest. F.B., D.M.,N.L., S.S., P.A.H., R.B., M.R., K.T.S.,K.D.R.R., S.Z.-E., M.B., V.F., S.T.,M.S. and M.H. are inventors on a patent application on blocking antibodies against SARS-CoV-2. A.H., A.F. and T.S. are officers of CORAT Therapeutics GmbH, a company founded by YUMAB GmbH for the clinical and regulatory development of STE90-C11 (COR-101). A.F., T.S., S.D. and M.H. are shareholders of YUMAB GmbH. Ethical Approval: Approval number, FV-2020-02. Approved by the TU Braunschweig Institute for Psychology.

Cell reports 36(4), 109433 (2021). doi:10.1016/j.celrep.2021.109433, The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC$_{50}$ in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 �� resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with Fc��R-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19., Published by Elsevier, [New York, NY]