{"doi":"10.1073/pnas.2216612120","title":"Fully synthetic platform to rapidly generate tetravalent bispecific nanobody–based immunoglobulins","abstract":"Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human V H 3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor–binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the V H and V L domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.","journal":"Proceedings of the National Academy of Sciences","year":2023,"id":323666,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":36,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9495,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":108970,"name":"Hejun Liu","orcid":null,"position":1,"is_corresponding":false},{"id":230741,"name":"Gabriel Ozorowski","orcid":"0000-0002-9695-8138","position":2,"is_corresponding":false},{"id":934132,"name":"Quoc Tran","orcid":"0000-0002-1250-5853","position":3,"is_corresponding":false},{"id":550937,"name":"Jordan L. Woehl","orcid":"0000-0001-7374-0059","position":4,"is_corresponding":false},{"id":983644,"name":"Irene S. Khalek","orcid":"0009-0000-3181-0434","position":5,"is_corresponding":false},{"id":1038951,"name":"Jessica Smith","orcid":"0009-0000-1554-6768","position":6,"is_corresponding":false},{"id":649569,"name":"Shawn Barman","orcid":"0000-0003-0008-2811","position":7,"is_corresponding":false},{"id":108971,"name":"Fangzhu Zhao","orcid":"0000-0002-3172-1340","position":8,"is_corresponding":false},{"id":523831,"name":"Celina Keating","orcid":"0000-0002-1314-9774","position":9,"is_corresponding":false},{"id":550935,"name":"Oliver Limbo","orcid":"0000-0003-1686-1414","position":10,"is_corresponding":false},{"id":1038952,"name":"Megan Verma","orcid":"0009-0000-4564-8132","position":11,"is_corresponding":false},{"id":915730,"name":"Jingjia Liu","orcid":"0000-0002-6038-593X","position":12,"is_corresponding":false},{"id":459198,"name":"Robyn L. Stanfield","orcid":"0000-0001-9521-9184","position":13,"is_corresponding":false},{"id":105425,"name":"Xueyong Zhu","orcid":"0000-0002-6021-3740","position":14,"is_corresponding":false},{"id":230742,"name":"Hannah L. Turner","orcid":"0000-0002-4745-8057","position":15,"is_corresponding":false},{"id":108978,"name":"Devin Sok","orcid":"0000-0002-9354-1740","position":16,"is_corresponding":false},{"id":317333,"name":"Po‐Ssu Huang","orcid":"0000-0002-7948-2895","position":17,"is_corresponding":false},{"id":108980,"name":"Dennis R. Burton","orcid":"0000-0001-6711-9864","position":18,"is_corresponding":false},{"id":230082,"name":"Andrew B. Ward","orcid":"0000-0001-7153-3769","position":19,"is_corresponding":false},{"id":105430,"name":"Ian A. Wilson","orcid":"0000-0002-6469-2419","position":20,"is_corresponding":false},{"id":108979,"name":"Joseph G. Jardine","orcid":"0000-0001-8951-1074","position":21,"is_corresponding":false},{"id":567016,"name":"Laëtitia Misson","orcid":"0000-0001-6318-1126","position":0,"is_corresponding":true}],"reference_count":51,"raw_metadata":null,"created_at":"2026-07-19T01:08:06.496579Z","pmid":"37276407","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}