--- _id: '12252' abstract: - lang: eng text: The COVID−19 pandemic not only resulted in a global crisis, but also accelerated vaccine development and antibody discovery. Herein we report a synthetic humanized VHH library development pipeline for nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) isolation. Trinucleotide-based randomization of CDRs by Kunkel mutagenesis with the subsequent rolling-cycle amplification resulted in more than 1011 diverse phage display library in a manageable for a single person number of electroporation reactions. We identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) by screening a novel synthetic humanized antibody library. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, these affinities are weakly influenced by most of SARS-CoV-2 VoC mutations and they retain moderate binding to BA.4\5. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and 9.6 ng\ml for H7-Fc and G12x3-Fc antibodies, respectively, for the emerging Omicron BA.1 variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants. acknowledgement: The authors declare that this study received funding from Immunofusion. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. article_number: '965446' article_processing_charge: No article_type: original author: - first_name: Dmitri full_name: Dormeshkin, Dmitri last_name: Dormeshkin - first_name: Michail full_name: Shapira, Michail last_name: Shapira - first_name: Simon full_name: Dubovik, Simon last_name: Dubovik - first_name: Anton full_name: Kavaleuski, Anton id: 4968f7ad-eb97-11eb-a6c2-8ed382e8912c last_name: Kavaleuski orcid: 0000-0003-2091-526X - first_name: Mikalai full_name: Katsin, Mikalai last_name: Katsin - first_name: Alexandr full_name: Migas, Alexandr last_name: Migas - first_name: Alexander full_name: Meleshko, Alexander last_name: Meleshko - first_name: Sergei full_name: Semyonov, Sergei last_name: Semyonov citation: ama: Dormeshkin D, Shapira M, Dubovik S, et al. Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library. Frontiers in Immunology. 2022;13. doi:10.3389/fimmu.2022.965446 apa: Dormeshkin, D., Shapira, M., Dubovik, S., Kavaleuski, A., Katsin, M., Migas, A., … Semyonov, S. (2022). Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library. Frontiers in Immunology. Frontiers Media. https://doi.org/10.3389/fimmu.2022.965446 chicago: Dormeshkin, Dmitri, Michail Shapira, Simon Dubovik, Anton Kavaleuski, Mikalai Katsin, Alexandr Migas, Alexander Meleshko, and Sergei Semyonov. “Isolation of an Escape-Resistant SARS-CoV-2 Neutralizing Nanobody from a Novel Synthetic Nanobody Library.” Frontiers in Immunology. Frontiers Media, 2022. https://doi.org/10.3389/fimmu.2022.965446. ieee: D. Dormeshkin et al., “Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library,” Frontiers in Immunology, vol. 13. Frontiers Media, 2022. ista: Dormeshkin D, Shapira M, Dubovik S, Kavaleuski A, Katsin M, Migas A, Meleshko A, Semyonov S. 2022. Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library. Frontiers in Immunology. 13, 965446. mla: Dormeshkin, Dmitri, et al. “Isolation of an Escape-Resistant SARS-CoV-2 Neutralizing Nanobody from a Novel Synthetic Nanobody Library.” Frontiers in Immunology, vol. 13, 965446, Frontiers Media, 2022, doi:10.3389/fimmu.2022.965446. short: D. Dormeshkin, M. Shapira, S. Dubovik, A. Kavaleuski, M. Katsin, A. Migas, A. Meleshko, S. Semyonov, Frontiers in Immunology 13 (2022). date_created: 2023-01-16T09:56:57Z date_published: 2022-09-16T00:00:00Z date_updated: 2023-08-04T09:49:24Z day: '16' ddc: - '570' department: - _id: LeSa doi: 10.3389/fimmu.2022.965446 external_id: isi: - '000862479100001' file: - access_level: open_access checksum: f8f5d8110710033d0532e7e08bf9dad4 content_type: application/pdf creator: dernst date_created: 2023-01-30T09:22:26Z date_updated: 2023-01-30T09:22:26Z file_id: '12443' file_name: 2022_FrontiersImmunology_Dormeshkin.pdf file_size: 5695892 relation: main_file success: 1 file_date_updated: 2023-01-30T09:22:26Z has_accepted_license: '1' intvolume: ' 13' isi: 1 keyword: - Immunology - Immunology and Allergy - COVID-19 - SARS-CoV-2 - synthetic library - RBD - neutralization nanobody - VHH language: - iso: eng month: '09' oa: 1 oa_version: Published Version publication: Frontiers in Immunology publication_identifier: issn: - 1664-3224 publication_status: published publisher: Frontiers Media quality_controlled: '1' scopus_import: '1' status: public title: Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library tmp: image: /images/cc_by.png legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0) short: CC BY (4.0) type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 13 year: '2022' ...