Language selection
- Personal Center
- Logout
FOLLOWUS
- Current Issue
- Current Issue
- Latest Articles
- Archive
- Special Issues
Your Location：
Home >
Browse articles >
Nanobodies in biomedicine: from molecular characteristics to fabrication and clinical translation
REVIEW | Updated：2026-05-11
|
Nanobodies in biomedicine: from molecular characteristics to fabrication and clinical translation
- Military Medical Research (2026)
- Affiliations：
School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, Zhejiang, China
National Engineering Research Center for Cell Growth Factor Drugs and Protein Biologics, Wenzhou 325000, Zhejiang, China
National Key Laboratory of Macromolecular Drug Development and Manufacturing, Wenzhou 325000, Zhejiang, China
- Author bio：
Xiao-Kun Li, xiaokunli@wmu.edu.cn
*Zhi-Jian Su, tjnuszj@jnu.edu.cn;
- Funds：
DOI：10.1016/j.mmr.2026.100009
CLC：Received：03 November 2025，
Accepted：06 March 2026，
Published：2026-04
- Accepted：
Scan QR Code
Cao CK, Xu XY, Liang Fei, Yao M, Chen YY, Li XK, et al. Nanobodies in biomedicine: from molecular characteristics to fabrication and clinical translation. Mil Med Res. 2026;13(1):100009.
Cao CK, Xu XY, Liang Fei, Yao M, Chen YY, Li XK, et al. Nanobodies in biomedicine: from molecular characteristics to fabrication and clinical translation. Mil Med Res. 2026;13(1):100009. DOI： 10.1016/j.mmr.2026.100009.
Nanobodies in biomedicine: from molecular characteristics to fabrication and clinical translation
摘要
Abstract
Nanobodies (Nbs)
the antigen-binding single-domain fragments derived from camelid heavy-chain antibodies (Abs)
have rapidly become a focus of biomedical research due to their compact size
high stability
strong antigen affinity
and ease of molecular engineering. This review systematically outlines their structural and functional features
current strategies for acquisition
screening
optimization
and large-scale production
and comprehensively discusses their wide-ranging applications in therapeutics
diagnostics
and basic research. Specifically
Nbs have shown outstanding efficacy in tumor
toxin
infectious
and cardiovascular disease treatments
while serving as versatile tools for molecular imaging
biosensing
protein purification
structural analysis
and intracellular regulation. The challenges of immunogenicity
off-target effects
and industrial-scale manufacturing are also critically examined. Furthermore
the integration of artificial intelligence in structure prediction
de novo design
and immunogenicity assessment has opened powerful new avenues for rational Nb engineering. Combined with emerging technologies such as gene therapy
nanomaterial delivery
and multispecific architectures
these advances promise to accelerate clinical translation. Overall
Nb technology is poised to become a cornerstone of next-generation precision medicine and biotechnology
offering innovative solutions for disease diagnosis
targeted therapy
and molecular discovery.
关键词
Keywords
references
Wang M , Ying T , Wu Y . Single-domain antibodies as therapeutics for solid tumor treatment . Acta Pharm Sin B . 2024 ; 14 ( 7 ): 2854 - 68 .
Koch‐Nolte F . Nanobody‐based heavy chain antibodies and chimeric antibodies . Immunol Rev . 2024 ; 328 ( 1 ): 466 - 72 .
Bao G , Tang M , Zhao J , Zhu X . Nanobody: a promising toolkit for molecular imaging and disease therapy . EJNMMI Res . 2021 ; 11 ( 1 ): 6 .
Blueggel M , Gül D , Stauber RH , Knauer SK . Small but mighty: the versatility of nanobodies in modern medicine . Nanoscale Horiz . 2025 ; 10 ( 10 ): 2158 - 71 .
Cheong WS , Leow CY , Abdul Majeed AB , Leow CH . Diagnostic and therapeutic potential of shark variable new antigen receptor (VNAR) single domain antibody . Int J Biol Macromol . 2020 ; 147 : 369 - 75 .
Rossotti MA , Bélanger K , Henry KA , Tanha J . Immunogenicity and humanization of single‐domain antibodies . FEBS J . 2022 ; 289 ( 14 ): 4304 - 27 .
Long X , Cheng S , Lan X , Wei W , Jiang D . Trends in nanobody radiotheranostics . Eur J Nucl Med Mol Imaging . 2025 ; 52 ( 6 ): 2225 - 38 .
Wu D , Xia S , Chen X . Nanobody‐based light‐controllable systems for investigating biology . ChemBioChem . 2025 ; 26 ( 14 ): e202500311 .
Ketaren NE , Fridy PC , Malashkevich V , Sanyal T , Brillantes M , Thompson MK , et al . Unique mechanisms to increase structural stability and enhance antigen binding in nanobodies . Structure . 2025 ; 33 ( 4 ): 677 - 90.e5 .
Shrestha P , Talwar CS , Kandel J , Park KH , Chong KT , Woo EJ , et al . NanoBinder: a machine learning assisted nanobody binding prediction tool using Rosetta energy scores . J Cheminform . 2025 ; 17 ( 1 ): 96 .
Ahmed FS , Aly S , El-Tabakh MAM , Liu X . NABP-LSTM-Att: Nanobody-antigen binding prediction using bidirectional LSTM and soft attention mechanism . Comput Biol Chem . 2025 ; 118 : 108490 .
Huo L , Tian T , Xu Y , Qin Q , Jiang X , Huang Q . Computational nanobody-design through deep generative modeling and epitope landscape profiling . Comput Struct Biotechnol J . 2025 ; 27 : 3443 - 55 .
Carter PJ , Rajpal A . Designing antibodies as therapeutics . Cell . 2022 ; 185 ( 15 ): 2789 - 805 .
Wilson IA , Stanfield RL . 50 years of structural immunology . J Biol Chem . 2021 ; 296 : 100745 .
Behring E , Kitasato S . Ueber das zustandekommen der diphtherie-immunität und der tetanus-immunität bei thieren . Dtsch Med Wochenschr . 1890 ; 49 : 1113 - 4 .
Raju TN . The nobel chronicles. 1972: Gerald M Edelman (b 1929) and Rodney R Porter (1917-85) . Lancet . 1999 ; 354 ( 9183 ): 1040 .
Jerne NK . The generative grammar of the immune system . Science . 1985 ; 229 ( 4718 ): 1057 - 9 .
Köhler G , Milstein C . Continuous cultures of fused cells secreting antibody of predefined specificity . Nature . 1975 ; 256 ( 5517 ): 495 - 7 .
Lu RM , Hwang YC , Liu IJ , Lee CC , Tsai HZ , Li HJ , et al . Development of therapeutic antibodies for the treatment of diseases . J Biomed Sci . 2020 ; 27 ( 1 ): 1 .
Crescioli S , Kaplon H , Wang L , Visweswaraiah J , Kapoor V , Reichert JM . Antibodies to watch in 2025 . MAbs . 2025 ; 17 ( 1 ): 2443538 .
Harris LJ , Larson SB , Hasel KW , McPherson A . Refined structure of an intact IgG2a monoclonal antibody . Biochemistry . 1997 ; 36 ( 7 ): 1581 - 97 .
Smith CA , Sonneson GJ , Hoey RJ , Hinerman JM , Sheehy K , Walter R , et al . Molecular recognition requires dimerization of a VHH antibody . MAbs . 2023 ; 15 ( 1 ): 2215363 .
Lou H , Cao X . Antibody variable region engineering for improving cancer immunotherapy . Cancer Commun . 2022 ; 42 ( 9 ): 804 - 27 .
Wang Z , Wang G , Lu H , Li H , Tang M , Tong A . Development of therapeutic antibodies for the treatment of diseases . Mol Biomed . 2022 ; 3 ( 1 ): 35 .
Vattepu R , Sneed SL , Anthony RM . Sialylation as an important regulator of antibody function . Front Immunol . 2022 ; 13 : 818736 .
Matsumoto ML . Molecular mechanisms of multimeric assembly of IgM and IgA . Annu Rev Immunol . 2022 ; 40 : 221 - 47 .
Rashid MH . Full-length recombinant antibodies from Escherichia coli : production, characterization, effector function (Fc) engineering, and clinical evaluation . MAbs . 2022 ; 14 ( 1 ): 2111748
Hamers-Casterman C , Atarhouch T , Muyldermans S , Robinson G , Hammers C , Songa EB , et al . Naturally occurring antibodies devoid of light chains . Nature . 1993 ; 363 ( 6428 ): 446 - 8 .
Jin B , Odongo S , Radwanska M , Magez S . NANOBODIES ® : a review of diagnostic and therapeutic applications . Int J Mol Sci . 2023 ; 24 ( 6 ): 5994 .
Hassanzadeh-Ghassabeh G , Devoogdt N , De Pauw P , Vincke C , Muyldermans S . Nanobodies and their potential applications . Nanomedicine . 2013 ; 8 ( 6 ): 1013 - 26 .
Fridy PC , Rout MP , Ketaren NE . Nanobodies: from high-throughput identification to therapeutic development . Mol Cell Proteomics . 2024 ; 23 ( 12 ): 100865 .
Yong Joon Kim J , Sang Z , Xiang Y , Shen Z , Shi Y . Nanobodies: robust miniprotein binders in biomedicine . Adv Drug Deliv Rev . 2023 ; 195 : 114726 .
Drugs@FDA: FDA-approved drugs – Cablivi (caplacizumab-yhdp) . U.S. Food and Drug Administration , Silver Spring (MD) . 2019 . https://www.accessdata.fda.gov/drugsatfda_docs/nda/2019/761112Orig1s000TOC.html https://www.accessdata.fda.gov/drugsatfda_docs/nda/2019/761112Orig1s000TOC.html . Accessed 26 Jan, 2026 .
Asaadi Y , Jouneghani FF , Janani S , Rahbarizadeh F . A comprehensive comparison between camelid nanobodies and single chain variable fragments . Biomark Res . 2021 ; 9 ( 1 ): 87 .
Moreno E , Valdés-Tresanco MS , Molina-Zapata A , Sánchez-Ramos O . Structure-based design and construction of a synthetic phage display nanobody library . BMC Res Notes . 2022 ; 15 ( 1 ): 124 .
Zhou P , Yang XL , Wang XG , Hu B , Zhang L , Zhang W , et al . A pneumonia outbreak associated with a new coronavirus of probable bat origin . Nature . 2020 ; 579 ( 7798 ): 270 - 3 .
Koenig PA , Das H , Liu H , Kümmerer BM , Gohr FN , Jenster LM , et al . Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape . Science . 2021 ; 371 ( 6530 ): eabe6230 .
King MT , Huh I , Shenai A , Brooks TM , Brooks CL . Structural basis of VHH-mediated neutralization of the food-borne pathogen Listeria monocytogenes . J Biol Chem . 2018 ; 293 ( 35 ): 13626 - 35 .
Braun MB , Traenkle B , Koch PA , Emele F , Weiss F , Poetz O , et al . Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy . Sci Rep . 2016 ; 6 : 19211 .
Noël F , Malpertuy A , de Brevern AG . Global analysis of VHHs framework regions with a structural alphabet . Biochimie . 2016 ; 131 : 11 - 9 .
Muyldermans S , Cambillau C , Wyns L . Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains . Trends Biochem Sci . 2001 ; 26 ( 4 ): 230 - 5 .
De Genst E , Saerens D , Muyldermans S , Conrath K . Antibody repertoire development in camelids . Dev Comp Immunol . 2006 ; 30 ( 1-2 ): 187 - 98 .
Schumacher D , Helma J , Schneider AFL , Leonhardt H , Hackenberger CPR . Nanobodies: chemical functionalization strategies and intracellular applications . Angew Chem Int Ed Engl . 2018 ; 57 ( 9 ): 2314 - 33 .
Yamamoto K , Nagatoishi S , Matsunaga R , Nakakido M , Kuroda D , Tsumoto K . Affinity‐stability trade‐off mechanism of residue 35 in framework region 2 of VHH antibodies with β‐hairpin CDR3 . Protein Sci . 2025 ; 34 ( 4 ): e70095 .
Murakami T , Kumachi S , Matsunaga Y , Sato M , Wakabayashi-Nakao K , Masaki H , et al . Construction of a humanized artificial VHH library reproducing structural features of camelid VHHs for therapeutics . Antibodies . 2022 ; 11 ( 1 ): 10 .
Muyldermans S . Nanobodies: natural single-domain antibodies . Annu Rev Biochem . 2013 ; 82 : 775 - 97 .
Xu M , Zhao Z , Deng P , Sun M , Chiu CKC , Wu Y , et al . Functional divergence in the affinity and stability of non-canonical cysteines and non-canonical disulfide bonds: insights from a VHH and VNAR study . Int J Mol Sci . 2024 ; 25 ( 18 ): 9801 .
Peng HP , Lee KH , Jian JW , Yang AS . Origins of specificity and affinity in antibody-protein interactions . Proc Natl Acad Sci U S A . 2014 ; 111 ( 26 ): E2656 - 65 .
Fernández-Quintero ML , Kokot J , Waibl F , Fischer ALM , Quoika PK , Deane CM , et al . Challenges in antibody structure prediction . MAbs . 2023 ; 15 ( 1 ): 2175319 .
Mitchell LS , Colwell LJ . Comparative analysis of nanobody sequence and structure data . Proteins . 2018 ; 86 ( 7 ): 697 - 706 .
Bannas P , Hambach J , Koch-Nolte F . Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics . Front Immunol . 2017 ; 8 : 1603 .
Bond CJ , Marsters JC , Sidhu SS . Contributions of CDR3 to VHH domain stability and the design of monobody scaffolds for naive antibody libraries . J Mol Biol . 2003 ; 332 ( 3 ): 643 - 55 .
Mendoza MN , Jian M , King MT , Brooks CL . Role of a noncanonical disulfide bond in the stability, affinity, and flexibility of a VHH specific for the Listeria virulence factor InlB . Protein Sci . 2020 ; 29 ( 4 ): 1004 - 1017 .
Martins C , Gardebien F , Nadaradjane AA , Diharce J , De Brevern AG . A simple analysis of the second (extra) disulfide bridge of VHHs . Molecules . 2024 ; 29 ( 20 ): 4863 .
Mitchell LS , Colwell LJ . Analysis of nanobody paratopes reveals greater diversity than classical antibodies . Protein Eng Des Sel . 2018 ; 31 ( 7-8 ): 267 - 75 .
Joubbi S , Micheli A , Milazzo P , Maccari G , Ciano G , Cardamone D , et al . Antibody design using deep learning: from sequence and structure design to affinity maturation . Brief Bioinform . 2024 ; 25 ( 4 ): bbae307 .
Liu C , Lin H , Cao L , Wang K , Sui J . Research progress on unique paratope structure, antigen binding modes, and systematic mutagenesis strategies of single-domain antibodies . Front Immunol . 2022 ; 13 : 1059771 .
Akiba H , Tamura H , Kiyoshi M , Yanaka S , Sugase K , Caaveiro JMM , et al . Structural and thermodynamic basis for the recognition of the substrate-binding cleft on hen egg lysozyme by a single-domain antibody . Sci Rep . 2019 ; 9 ( 1 ): 15481 .
Barakat S , Berksöz M , Zahedimaram P , Piepoli S , Erman B . Nanobodies as molecular imaging probes . Free Radic Biol Med . 2022 ; 182 : 260 - 75 .
Kunz S , Durandy M , Seguin L , Feral CC . NANOBODY ® molecule, a giga medical tool in nanodimensions . Int J Mol Sci . 2023 ; 24 ( 17 ): 13229 .
Li Q , Zhang F , Lu Y , Hu H , Wang J , Guo C , et al . Highly potent multivalent VHH antibodies against chikungunya isolated from an alpaca naïve phage display library . J Nanobiotechnology . 2022 ; 20 ( 1 ): 231 .
Sockolosky JT , Dougan M , Ingram JR , Ho CCM , Kauke MJ , Almo SC , et al . Durable antitumor responses to CD47 blockade require adaptive immune stimulation . Proc Natl Acad Sci U S A . 2016 ; 113 ( 19 ): E2646 - 54 .
Rothbauer U , Zolghadr K , Muyldermans S , Schepers A , Cardoso MC , Leonhardt H . A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins . Mol Cell Proteomics . 2008 ; 7 ( 2 ): 282 - 9 .
Kubala MH , Kovtun O , Alexandrov K , Collins BM . Structural and thermodynamic analysis of the GFP:GFP‐nanobody complex . Protein Sci . 2010 ; 19 ( 12 ): 2389 - 401 .
Dumoulin M , Conrath K , Van Meirhaeghe A , Meersman F , Heremans K , Frenken LGJ , et al . Single‐domain antibody fragments with high conformational stability . Protein Sci . 2002 ; 11 ( 3 ): 500 - 15 .
Alexander E , Leong KW . Discovery of nanobodies: a comprehensive review of their applications and potential over the past five years . J Nanobiotechnology . 2024 ; 22 ( 1 ): 661 .
Olichon A , Schweizer D , Muyldermans S , De Marco A . Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains . BMC Biotechnol . 2007 ; 7 : 7 .
Hussack G , Hirama T , Ding W , MacKenzie R , Tanha J . Engineered single-domain antibodies with high protease resistance and thermal stability . PLoS One . 2011 ; 6 ( 11 ): e28218 .
Ma H , Ó’Fágáin C , O’Kennedy R . Antibody stability: a key to performance-analysis, influences and improvement . Biochimie . 2020 ; 177 : 213 - 25 .
Bélanger K , Wu C , Sulea T , Van Faassen H , Callaghan D , Aubry A , et al . Optimization of synthetic human VH affinity and solubility through in vitro affinity maturation and minimal camelization . Protein Sci . 2025 ; 34 ( 5 ): e70114 .
Uto Y , Nakakido M , Yokoo T , Fernandez‐Perez J , Entzminger K , Maruyama T , et al . Improving the solubility of single domain antibodies using VH-like hallmark residues . Protein Sci . 2025 ; 34 ( 7 ): e70189 .
Abdeldaim DT , Schindowski K . Fc-engineered therapeutic antibodies: recent advances and future directions . Pharmaceutics . 2023 ; 15 ( 10 ): 2402 .
Wilkinson I , Anderson S , Fry J , Julien LA , Neville D , Qureshi O , et al . Fc-engineered antibodies with immune effector functions completely abolished . PLoS One . 2021 ; 16 ( 12 ): e0260954 .
Ackaert C , Smiejkowska N , Xavier C , Sterckx YGJ , Denies S , Stijlemans B , et al . Immunogenicity risk profile of nanobodies . Front Immunol . 2021 ; 12 : 632687 .
Ewert S , Cambillau C , Conrath K , Plückthun A . Biophysical properties of camelid V(HH) domains compared to those of human V(H)3 domains . Biochemistry . 2002 ; 41 ( 11 ): 3628 - 36 .
Lee NJ , Jung M , Yang HY , Shim H . A single-domain antibody library based on a stability-engineered human VH3 scaffold . Sci Rep . 2024 ; 14 ( 1 ): 17747 .
Klarenbeek A , El Mazouari K , Desmyter A , Blanchetot C , Hultberg A , de Jonge N , et al . Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform . MAbs . 2015 ; 7 ( 4 ): 693 - 706 .
Aoki K , Manabe A , Kimura H , Katoh Y , Inuki S , Ohno H , et al . Mirror-image single-domain antibody for a novel nonimmunogenic drug scaffold . Bioconjug Chem . 2023 ; 34 ( 11 ): 2055 - 65 .
Aoki K , Higashi K , Oda S , Manabe A , Maeda K , Morise J , et al . Engineering a low-immunogenic mirror-image VHH against vascular endothelial growth factor . ACS Chem Biol . 2024 ; 19 ( 5 ): 1194 - 205 .
Hartmann L , Botzanowski T , Galibert M , Jullian M , Chabrol E , Zeder‐Lutz G , et al . VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH . Protein Sci . 2019 ; 28 ( 10 ): 1865 - 79 .
Oosterlaken M , Rogliardo A , Lipina T , Lafon PA , Tsitokana ME , Keck M , et al . Nanobody therapy rescues behavioural deficits of NMDA receptor hypofunction . Nature . 2025 ; 645 ( 8079 ): 262 - 70 .
Ruiz-López E , Schuhmacher AJ . Transportation of single-domain antibodies through the blood-brain barrier . Biomolecules . 2021 ; 11 ( 8 ): 1131 .
Pothin E , Lesuisse D , Lafaye P . Brain delivery of single-domain antibodies: a focus on VHH and VNAR . Pharmaceutics . 2020 ; 12 ( 10 ): 937 .
Ma X , Liu T , Guo R , Zhou W , Yao Y , Wen D , et al . Radioiodinated nanobody immunoPET probe for in vivo detection of CD147 in pan-cancer . Eur J Nucl Med Mol Imaging . 2025 ; 52 ( 4 ): 1406 - 21 .
Eyssen LE- , Ramadurai S , Abdelkarim S , Buckle I , Cornish K , Lin H , et al . From llama to nanobody: a streamlined workflow for the generation of functionalised VHHs . Bio Protoc . 2024 ; 14 ( 6 ): e4962 .
Minatel VM , Prudencio CR , Barraviera B , Ferreira RS . Nanobodies: a promising approach to treatment of viral diseases . Front Immunol . 2024 ; 14 : 1303353 .
Liu W , Song H , Chen Q , Yu J , Xian M , Nian R , et al . Recent advances in the selection and identification of antigen-specific nanobodies . Mol Immunol . 2018 ; 96 : 37 - 47 .
Rossotti MA , Trempe F , van Faassen H , Hussack G , Arbabi-Ghahroudi M . Isolation and characterization of single-domain antibodies from immune phage display libraries . Methods Mol Biol . 2023 ; 2702 : 107 - 147 .
Muyldermans S . A guide to: generation and design of nanobodies . FEBS J . 2021 ; 288 ( 7 ): 2084 - 102 .
Qiu J , Li J , Zhang Z , Dong S , Ling X , Fang Z , et al . Construction of an alpaca immune antibody library for the selection of nanobodies against Drosophila melanogaster proteins . Front Bioeng Biotechnol . 2023 ; 11 : 1207048 .
Wu X , Zhu L , Liang S , Chang Z , Wang Y , Zou Q , et al . A rationally designed cocktail of nanobodies elicited by heterologous vaccination confers protection against SFTSV in preclinical models . Sci Transl Med . 2025 ; 17 ( 825 ): eady9025 .
Eden T , Schaffrath AZ , Wesolowski J , Stähler T , Tode N , Richter N , et al . Generation of nanobodies from transgenic ‘LamaMice’ lacking an endogenous immunoglobulin repertoire . Nat Commun . 2024 ; 15 ( 1 ): 4728 .
Lu Q , Zhang Z , Li H , Zhong K , Zhao Q , Wang Z , et al . Development of multivalent nanobodies blocking SARS-CoV-2 infection by targeting RBD of spike protein . J Nanobiotechnology . 2021 ; 19 ( 1 ): 33 .
Wang M , Wei L , Xiang H , Ren B , Liu X , Jiang L , et al . A megadiverse naïve library derived from numerous camelids for efficient and rapid development of VHH antibodies . Anal Biochem . 2022 ; 657 : 114871 .
Lee HE , Cho AH , Hwang JH , Kim JW , Yang HR , Ryu T , et al . Development, high-throughput profiling, and biopanning of a large phage display single-domain antibody library . Int J Mol Sci . 2024 ; 25 ( 9 ): 4791 .
Mikolajek H , Weckener M , Brotzakis ZF , Huo J , Dalietou EV , Le Bas A , et al . Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes . Proc Natl Acad Sci U S A . 2022 ; 119 ( 31 ): e2205412119 .
Valdés-Tresanco MS , Molina-Zapata A , Pose AG , Moreno E . Structural insights into the design of synthetic nanobody libraries . Molecules . 2022 ; 27 ( 7 ): 2198 .
Chen F , Ma H , Li Y , Wang H , Samad A , Zhou J , et al . Screening of nanobody specific for peanut major allergen Ara h 3 by phage display . J Agric Food Chem . 2019 ; 67 ( 40 ): 11219 - 29 .
Dormeshkin D , Shapira M , Dubovik S , Kavaleuski A , Katsin M , Migas A , et al . Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library . Front Immunol . 2022 ; 13 : 965446 .
Kim M , Bai X , Im H , Yang J , Kim Y , Kim MM , et al . Construction and validation of a synthetic phage-displayed nanobody library . Korean J Physiol Pharmacol . 2024 ; 28 ( 5 ): 457 - 67 .
Wagner HJ , Wehrle S , Weiss E , Cavallari M , Weber W . A two-step approach for the design and generation of nanobodies . Int J Mol Sci . 2018 ; 19 ( 11 ): 3444 .
Aguilar Rangel M , Bedwell A , Costanzi E , Taylor RJ , Russo R , Bernardes GJL , et al . Fragment-based computational design of antibodies targeting structured epitopes . Sci Adv . 2022 ; 8 ( 45 ): eabp9540 .
Liu B , Yang D . Easily established and multifunctional synthetic nanobody libraries as research tools . Int J Mol Sci . 2022 ; 23 ( 3 ): 1482 .
Contreras MA , Serrano-Rivero Y , González-Pose A , Salazar-Uribe J , Rubio-Carrasquilla M , Soares-Alves M , et al . Design and construction of a synthetic nanobody library: testing its potential with a single selection round strategy . Molecules . 2023 ; 28 ( 9 ): 3708 .
Zimmermann I , Egloff P , Hutter CAJ , Kuhn BT , Bräuer P , Newstead S , et al . Generation of synthetic nanobodies against delicate proteins . Nat Protoc . 2020 ; 15 ( 5 ): 1707 - 41 .
Roth KDR , Wenzel EV , Ruschig M , Steinke S , Langreder N , Heine PA , et al . Developing recombinant antibodies by phage display against infectious diseases and toxins for diagnostics and therapy . Front Cell Infect Microbiol . 2021 ; 11 : 697876 .
Ledsgaard L , Ljungars A , Rimbault C , Sørensen CV , Tulika T , Wade J , et al . Advances in antibody phage display technology . Drug Discov Today . 2022 ; 27 ( 8 ): 2151 - 69 .
Mustafa MI , Mohammed A . Developing recombinant antibodies by phage display technology to neutralize viral infectious diseases . SLAS Discov . 2024 ; 29 ( 3 ): 100140 .
Guliy OI , Evstigneeva SS , Dykman LA . Recombinant antibodies by phage display for bioanalytical applications . Biosens Bioelectron . 2023 ; 222 : 114909 .
Lu J , Ding J , Liu Z , Chen T . Retrospective analysis of the preparation and application of immunotherapy in cancer treatment (Review) . Int J Oncol . 2022 ; 60 ( 2 ): 12 .
Mahdavi SZB , Oroojalian F , Eyvazi S , Hejazi M , Baradaran B , Pouladi N , et al . An overview on display systems (phage, bacterial, and yeast display) for production of anticancer antibodies; advantages and disadvantages . Int J Biol Macromol . 2022 ; 208 : 421 - 42 .
John P , Sriram S , Palanichamy C , Subash PT , Sudandiradoss C . A multifarious bacterial surface display: potential platform for biotechnological applications . Crit Rev Microbiol . 2025 ; 51 ( 6 ): 1015 - 1040 .
Xiang HY , Wang JF , Yang YF , Lu YC . The research progress of bacterial surface display technology . Prog Biochem Biophys . 2019 ; 46 ( 2 ): 162 - 8. in Chinese .
Salema V , Fernández LÁ . Escherichia coli surface display for the selection of nanobodies . Microb Biotechnol . 2017 ; 10 ( 6 ): 1468 - 84 .
Liu W , Sun W , Liang C , Chen T , Zhuang W , Liu D , et al . Escherichia coli surface display: advances and applications in biocatalysis . ACS Synth Biol . 2025 ; 14 ( 3 ): 648 - 61 .
Dahlsson Leitao C , Ståhl S , Löfblom J . Surface‐engineered bacteria in drug development . Microb Biotechnol . 2024 ; 17 ( 10 ): e70033 .
Bahrulolum H , Ahmadian G . Bacillus subtilis surface display technology: applications in bioprocessing and sustainable manufacturing . Biotechnol Biofuels Bioprod . 2025 ; 18 ( 1 ): 34 .
Li Y , Wang X , Zhou NY , Ding J . Yeast surface display technology: mechanisms, applications, and perspectives . Biotechnol Adv . 2024 ; 76 : 108422 .
Xing H , Zhu L , Wang P , Zhao G , Zhou Z , Yang Y , et al . Display of receptor-binding domain of SARS-CoV-2 spike protein variants on the Saccharomyces cerevisiae cell surface . Front Immunol . 2022 ; 13 : 935573 .
Yang X , Tang H , Song M , Shen Y , Hou J , Bao X . Development of novel surface display platforms for anchoring heterologous proteins in Saccharomyces cerevisiae . Microb Cell Fact . 2019 ; 18 ( 1 ): 85 .
Zhang C , Chen H , Zhu Y , Zhang Y , Li X , Wang F . Saccharomyces cerevisiae cell surface display technology: strategies for improvement and applications . Front Bioeng Biotechnol . 2022 ; 10 : 1056804 .
Slavny P , Hegde M , Doerner A , Parthiban K , McCafferty J , Zielonka S , et al . Advancements in mammalian display technology for therapeutic antibody development and beyond: current landscape, challenges, and future prospects . Front Immunol . 2024 ; 15 : 1469329 .
Valldorf B , Hinz SC , Russo G , Pekar L , Mohr L , Klemm J , et al . Antibody display technologies: selecting the cream of the crop . Biol Chem . 2021 ; 403 ( 5-6 ): 455 - 77 .
Nakai H , Isshiki K , Hattori M , Maehira H , Yamaguchi T , Masuda K , et al . Cell-free synthesis of human endothelin receptors and its application to ribosome display . Anal Chem . 2022 ; 94 ( 9 ): 3831 - 9 .
Nemoto N , Fukushima T , Kumachi S , Suzuki M , Nishigaki K , Kubo T . Versatile C-terminal specific biotinylation of proteins using both a puromycin-linker and a cell-free translation system for studying high-throughput protein-molecule interactions . Anal Chem . 2014 ; 86 ( 17 ): 8535 - 40 .
Kunamneni A , Ogaugwu C , Bradfute S , Durvasula R . Ribosome display technology: applications in disease diagnosis and control . Antibodies . 2020 ; 9 ( 3 ): 28 .
Kamalinia G , Grindel BJ , Takahashi TT , Millward SW , Roberts RW . Directing evolution of novel ligands by mRNA display . Chem Soc Rev . 2021 ; 50 ( 16 ): 9055 - 103 .
Wyrzucki A , Dreyfus C , Kohler I , Steck M , Wilson IA , Hangartner L . Alternative recognition of the conserved stem epitope in influenza A virus hemagglutinin by a VH3-30-encoded heterosubtypic antibody . J Virol . 2014 ; 88 ( 12 ): 7083 - 92 .
Hägglöf T , Cipolla M , Loewe M , Chen ST , Kara EE , Mesin L , et al . Continuous germinal center invasion contributes to the diversity of the immune response . Cell . 2023 ; 186 ( 1 ): 147 - 161.e15 .
Sulea T , Baardsnes J , Stuible M , Rohani N , Tran A , Parat M , et al . Structure-based dual affinity optimization of a SARS-CoV-1/2 cross-reactive single-domain antibody . PLoS One . 2022 ; 17 ( 3 ): e0266250 .
Ribeiro R , Vítor JMB , Voronovska A , Moreira JN , Goncalves J . Novel strategy of antibody affinity maturation and enhancement of nucleolin‐mediated antibody‐dependent cellular cytotoxicity against triple‐negative breast cancer . Biotechnol J . 2025 ; 20 ( 1 ): e202400380 .
Hoseinpoor R , Mousavi Gargari SL , Rasooli I , Rajabibazl M , Shahi B . Functional mutations in and characterization of VHH against Helicobacter pylori urease . Appl Biochem Biotechnol . 2014 ; 172 ( 6 ): 3079 - 91 .
Li J , Kang G , Wang J , Yuan H , Wu Y , Meng S , et al . Affinity maturation of antibody fragments: a review encompassing the development from random approaches to computational rational optimization . Int J Biol Macromol . 2023 ; 247 : 125733 .
Hameduh T , Haddad Y , Adam V , Heger Z . Homology modeling in the time of collective and artificial intelligence . Comput Struct Biotechnol J . 2020 ; 18 : 3494 - 506 .
Guest JD , Vreven T , Zhou J , Moal I , Jeliazkov JR , Gray JJ , et al . An expanded benchmark for antibody-antigen docking and affinity prediction reveals insights into antibody recognition determinants . Structure . 2021 ; 29 ( 6 ): 606 - 21.e5 .
Sun Y , Jiao Y , Shi C , Zhang Y . Deep learning-based molecular dynamics simulation for structure-based drug design against SARS-CoV-2 . Comput Struct Biotechnol J . 2022 ; 20 : 5014 - 27 .
Yoshida K , Kuroda D , Kiyoshi M , Nakakido M , Nagatoishi S , Soga S , et al . Exploring designability of electrostatic complementarity at an antigen-antibody interface directed by mutagenesis, biophysical analysis, and molecular dynamics simulations . Sci Rep . 2019 ; 9 ( 1 ): 4482 .
He T , Nie Y , Yan T , Zhu J , He X , Li Y , et al . Enhancing the detection sensitivity of nanobody against aflatoxin B1 through structure-guided modification . Int J Biol Macromol . 2022 ; 194 : 188 - 97 .
Cai H , Zhang Z , Wang M , Zhong B , Li Q , Zhong Y , et al . Pretrainable geometric graph neural network for antibody affinity maturation . Nat Commun . 2024 ; 15 ( 1 ): 7785 .
Yu H , Mao G , Pei Z , Cen J , Meng W , Wang Y , et al . In vitro affinity maturation of nanobodies against MPOX virus A29 protein based on computer-aided design . Molecules . 2023 ; 28 ( 19 ): 6838 .
Liang T , Sun ZY , Hines MG , Penrose KJ , Hao Y , Chu X , et al . AI-based IsAb2.0 for antibody design . Brief Bioinform . 2024 ; 25 ( 5 ): bbae445 .
Ikeuchi E , Kuroda D , Nakakido M , Murakami A , Tsumoto K . Delicate balance among thermal stability, binding affinity, and conformational space explored by single-domain VHH antibodies . Sci Rep . 2021 ; 11 ( 1 ): 20624 .
Li J , Deng Y , Zhang W , Zhou AP , Guo W , Yang J , et al . Subcutaneous envafolimab monotherapy in patients with advanced defective mismatch repair/microsatellite instability high solid tumors . J Hematol Oncol . 2021 ; 14 ( 1 ): 95 .
Papadopoulos KP , Harb W , Peer CJ , Hua Q , Xu S , Lu H , et al . First-in-human phase Ⅰ study of envafolimab, a novel subcutaneous single-domain anti-PD-L1 antibody, in patients with advanced solid tumors . Oncologist . 2021 ; 26 ( 9 ): e1514 - 25 .
Giraudet R , Laroche A , Chalopin B , Dubois S , Correia E , Staropoli I , et al . Immunogenicity of single-chain antibodies: germlining of a VHH lowers T-cell activation from epitopes in FR2 and CDR regions . MAbs . 2025 ; 17 ( 1 ): 2571406 .
Sulea T . Humanization of camelid single-domain antibodies . Methods Mol Biol . 2022 ; 2446 : 299 - 312 .
Marino SF , Daumke O . Structure-based humanization of a therapeutic antibody for multiple myeloma . J Mol Med . 2024 ; 102 ( 9 ): 1151 - 61 .
Vincke C , Loris R , Saerens D , Martinez-Rodriguez S , Muyldermans S , Conrath K . General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold . J Biol Chem . 2009 ; 284 ( 5 ): 3273 - 84 .
Kurella VB , Gali R . Antibody design and humanization via in silico modeling . Methods Mol Biol . 2018 ; 1827 : 3 - 14 .
Wang Y , Chen YL , Xu H , Rana GE , Tan X , He M , et al . Comparison of “framework Shuffling” and “CDR Grafting” in humanization of a PD-1 murine antibody . Front Immunol . 2024 ; 15 : 1395854 .
Gordon GL , Raybould MIJ , Wong A , Deane CM . Prospects for the computational humanization of antibodies and nanobodies . Front Immunol . 2024 ; 15 : 1399438 .
Amash A , Volkers G , Farber P , Griffin D , Davison KS , Goodman A , et al . Developability considerations for bispecific and multispecific antibodies . MAbs . 2024 ; 16 ( 1 ): 2394229 .
Oostindie SC , Lazar GA , Schuurman J , Parren PWHI . Avidity in antibody effector functions and biotherapeutic drug design . Nat Rev Drug Discov . 2022 ; 21 ( 10 ): 715 - 35 .
Bachmann MF , Krenger PS , Mohsen MO , Kramer MF , Starchenka S , Whitehead P , et al . On the role of antibody affinity and avidity in the IgE ‐mediated allergic response . Allergy . 2025 ; 80 ( 1 ): 37 - 46 .
Mullin M , McClory J , Haynes W , Grace J , Robertson N , Van Heeke G . Applications and challenges in designing VHH-based bispecific antibodies: leveraging machine learning solutions . MAbs . 2024 ; 16 ( 1 ): 2341443 .
Wang J , Kang G , Yuan H , Cao X , Huang H , De Marco A . Research progress and applications of multivalent, multispecific and modified nanobodies for disease treatment . Front Immunol . 2022 ; 12 : 838082 .
Ding Z , Sun S , Wang X , Yang X , Shi W , Huang X , et al . Nanobody-based trispecific T cell engager (Nb-TriTE) enhances therapeutic efficacy by overcoming tumor-mediated immunosuppression . J Hematol Oncol . 2023 ; 16 ( 1 ): 115 .
Hanke L , Das H , Sheward DJ , Perez Vidakovics L , Urgard E , Moliner-Morro A , et al . A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo . Nat Commun . 2022 ; 13 ( 1 ): 155 .
Al-Seragi M , Chen Y , Duong van Hoa F . Advances in nanobody multimerization and multispecificity: from in vivo assembly to in vitro production . Biochem Soc Trans . 2025 ; 53 ( 1 ): 235 - 48 .
Misson Mindrebo L , Liu H , Ozorowski G , Tran Q , Woehl J , Khalek I , et al . Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins . Proc Natl Acad Sci U S A . 2023 ; 120 ( 24 ): e2216612120 .
Wu T , Huang H , Sheng Y , Shi H , Min Y , Liu Y . Transglutaminase mediated PEGylation of nanobodies for targeted nano-drug delivery . J Mater Chem B . 2018 ; 6 ( 7 ): 1011 - 7 .
Wu Y , Zhu M , Sun B , Chen Y , Huang Y , Gai J , et al . A humanized trivalent Nectin-4-targeting nanobody drug conjugate displays potent antitumor activity in gastric cancer . J Nanobiotechnology . 2024 ; 22 ( 1 ): 256 .
Nguyen TD , Bordeau BM , Zhang Y , Mattle AG , Balthasar JP . Half-life extension and biodistribution modulation of biotherapeutics via red blood cell hitch-hiking with novel anti-band 3 single-domain antibodies . Int J Mol Sci . 2022 ; 24 ( 1 ): 475 .
Cuypers ML , Jaspers T , Clerckx J , Leekens S , Cawthorne C , Bormans G , et al . Increasing brain half-life of antibodies by additional binding to myelin oligodendrocyte glycoprotein, a CNS specific protein . Fluids Barriers CNS . 2025 ; 22 ( 1 ): 11 .
Liu J , Hong H , Shi J , Xie Y , Lu Z , Liu Z , et al . Dinitrophenol-mediated modulation of an anti-PD-L1 VHH for Fc-dependent effector functions and prolonged serum half-life . Eur J Pharm Sci . 2021 ; 165 : 105941 .
Hong H , Zhou Z , Zhou K , Liu S , Guo Z , Wu Z . Site-specific C-terminal dinitrophenylation to reconstitute the antibody Fc functions for nanobodies . Chem Sci . 2019 ; 10 ( 40 ): 9331 - 8 .
Fan K , Jiang B , Guan Z , He J , Yang D , Xie N , et al . Fenobody: a ferritin-displayed nanobody with high apparent affinity and half-life extension . Anal Chem . 2018 ; 90 ( 9 ): 5671 - 7 .
Chiani M , Abedini R , Ahangari-Cohan R , Behdani M , Barzi SM , Mohseni N , et al . Encapsulation of anti-VEGF nanobody into niosome nanoparticles: a novel approach to enhance circulation half life and efficacy . J Microencapsul . 2025 ; 42 ( 2 ): 132 - 41 .
de Marco A . Recombinant expression of nanobodies and nanobody-derived immunoreagents . Protein Expr Purif . 2020 ; 172 : 105645 .
Tungekar AA , Castillo-Corujo A , Ruddock LW . So you want to express your protein in Escherichia coli ? . Essays Biochem . 2021 ; 65 ( 2 ): 247 - 60 .
Castillo-Corujo A , Uchida Y , Saaranen MJ , Ruddock LW . Escherichia coli cytoplasmic expression of disulfide-bonded proteins: side-by-side comparison between two competing strategies . J Microbiol Biotechnol . 2024 ; 34 ( 5 ): 1126 - 34 .
Manta B , Boyd D , Berkmen M . Disulfide bond formation in the periplasm of Escherichia coli . EcoSal Plus . 2019 ; 8 ( 2 ): 10.1128/ecosalplus.ESP-0012-2018 https://doi.org/10.1128/ecosalplus.ESP-0012-2018 .
Maggi M , Scotti C . Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies . Protein Expr Purif . 2017 ; 136 : 39 - 44 .
Hao Z , Dong X , Zhang Z , Qin Z . A nanobody of PEDV S1 protein: screening and expression in Escherichia coli . Biomolecules . 2024 ; 14 ( 19 ): 1116 .
Yao G , Huang C , Ji F , Ren J , Luo X , Zang B , et al . Nanobodies as solubilization chaperones for the expression and purification of inclusion-body prone proteins . Chem Commun . 2022 ; 58 ( 17 ): 2898 - 901 .
Billen B , Vincke C , Hansen R , Devoogdt N , Muyldermans S , Adriaensens P , et al . Cytoplasmic versus periplasmic expression of site-specifically and bioorthogonally functionalized nanobodies using expressed protein ligation . Protein Expr Purif . 2017 ; 133 : 25 - 34 .
Katiyar R , Gindi N , Mittal I , Komives C , Rathore AS . Vitamin supplementation for enhanced production of therapeutic nanobody in E. coli . Biochem Eng J . 2025 ; 223 : 109887 .
Sandomenico A , Sivaccumar JP , Ruvo M . Evolution of Escherichia coli expression system in producing antibody recombinant fragments . Int J Mol Sci . 2020 ; 21 ( 17 ): 6324 .
Zhao S , Zeng W , Yu F , Xu P , Chen CY , Chen W , et al . Visual and high-efficiency secretion of SARS-CoV-2 nanobodies with Escherichia coli . Biomolecules . 2025 ; 15 ( 1 ): 111 .
Kavousipour S , Mohammadi S , Eftekhar E , Barazesh M , Morowvat MH . In silico investigation of signal peptide sequences to enhance secretion of CD44 nanobodies expressed in Escherichia coli . Curr Pharm Biotechnol . 2021 ; 22 ( 9 ): 1192 - 205 .
Lynch JP , González-Prieto C , Reeves AZ , Bae S , Powale U , Godbole NP , et al . Engineered Escherichia coli for the in situ secretion of therapeutic nanobodies in the gut . Cell Host Microbe . 2023 ; 31 ( 4 ): 634 - 649.e8 .
İncir İ , Kaplan Ö . Escherichia coli in the production of biopharmaceuticals . Biotechnol Appl Biochem . 2025 ; 72 ( 2 ): 528 - 41 .
Matsuzaki Y , Kajiwara K , Aoki W , Ueda M . Production of single-domain antibodies in Pichia pastoris . Methods Mol Biol . 2022 ; 2446 : 181 - 203 .
Karbalaei M , Rezaee SA , Farsiani H . Pichia pastoris : a highly successful expression system for optimal synthesis of heterologous proteins . J Cell Physiol . 2020 ; 235 ( 9 ): 5867 - 81 .
Zhou Z , Liao B , Wang S , Tang J , Zhao H , Tong M , et al . Improved production of anti-FGF-2 nanobody using Pichia pastoris and its effect on antiproliferation of keratinocytes and alleviation of psoriasis . Arch Immunol Ther Exp . 2023 ; 71 ( 1 ): 20 .
Unver Y , Dagci I . Komagataella phaffii ( Pichia pastoris ) as a powerful yeast expression system for biologics production . Front Biosci . 2024 ; 16 ( 2 ): 19 .
Eskandari A , Nezhad NG , Leow TC , Rahman MBA , Oslan SN . Current achievements, strategies, obstacles, and overcoming the challenges of the protein engineering in Pichia pastoris expression system . World J Microbiol Biotechnol . 2023 ; 40 ( 1 ): 39 .
Xue A , Fan S . Matrices and affinity ligands for antibody purification and corresponding applications in radiotherapy . Biomolecules . 2022 ; 12 ( 6 ): 821 .
Yang X , Chen B , Lao Z , Xiang Y , Lin Z . A spy chemistry-based method for purification of proteins with authentic N-termini . Catalysts . 2024 ; 14 ( 9 ): 651 .
Iwaki T , Hara K , Umemura K . Nanobody production can be simplified by direct secretion from Escherichia coli . Protein Expr Purif . 2020 ; 170 : 105607 .
Joucla G , Le Sénéchal C , Bégorre M , Garbay B , Santarelli X , Cabanne C . Cation exchange versus multimodal cation exchange resins for antibody capture from CHO supernatants: identification of contaminating host cell proteins by mass spectrometry . J Chromatogr B Analyt Technol Biomed Life Sci . 2013 ; 942-943 : 126 - 33 .
Li X , Liu W , Li H , Wang X , Zhao Y . Capture and purification of an untagged nanobody by mixed weak cation chromatography and cation exchange chromatography . Protein Expr Purif . 2022 ; 192 : 106030 .
Berg EA , Fishman JB . Purification of labeled antibodies using size-exclusion chromatography . Cold Spring Harb Protoc . 2020 ; 2020 ( 7 ): 099358 .
Djulbegovic M , Tong J , Xu A , Yang J , Chen Y , Cuker A , et al . Adding caplacizumab to standard of care in thrombotic thrombocytopenic purpura: a systematic review and meta-analysis . Blood Adv . 2023 ; 7 ( 10 ): 2132 - 42 .
Markham A . Envafolimab: first approval . Drugs . 2022 ; 82 ( 2 ): 235 - 40 .
Jagannath S , Jackson CC , Schecter JM , Lendvai N , Sun H , Akram M , et al . Cilta-cel, a BCMA-targeting CAR-T therapy for patients with multiple myeloma . Expert Opin Biol Ther . 2024 ; 24 ( 5 ): 339 - 50 .
Keam SJ . Ozoralizumab: first approval . Drugs . 2023 ; 83 ( 1 :) 87 - 92 .
Papp KA , Weinberg MA , Morris A , Reich K . IL17A/F nanobody sonelokimab in patients with plaque psoriasis: a multicentre, randomised, placebo-controlled, phase 2b study . Lancet . 2021 ; 397 ( 10284 ): 1564 - 75 .
Jindal S , Pedersen DV , Gera N , Chandler J , Patel R , Neill A , et al . Characterization of the bispecific VHH antibody gefurulimab (ALXN1720) targeting complement component 5, and designed for low volume subcutaneous administration . Mol Immunol . 2024 ; 165 : 29 - 41 .
Alshrari AS , Hudu SA , Elmigdadi F , Imran M . The urgent threat of Clostridioides difficile infection: a glimpse of the drugs of the future, with related patents and prospects . Biomedicines . 2023 ; 11 ( 2 ): 426 .
Liu D , Gong J , Li J , Qi C , Niu Z , Liu B , et al . Efficacy and safety of KN026, a bispecific anti-HER2 antibody, in combination with KN046, an anti-CTLA4/PD-L1 antibody, in patients with advanced HER2-positive nonbreast cancer: a combined analysis of a phase Ib and a phase Ⅱ study . Signal Transduct Target Ther . 2025 ; 10 ( 1 ): 104 .
Leeuw T , Šimaitė D , Heyninck K , Levin C , Cornelis S , Hijazi Y , et al . Combined TNF-α and OX40L targeting as a new treatment option for hidradenitis suppurativa . J Allergy Clin Immunol Glob . 2025 ; 4 ( 3 ): 100483 .
Sinclair R , Sharifeh S , Thackwray S , Lickliter J , Wu J , Li J , et al . Topical application of a novel anti‐interleukin‐17A antibody fragment penetrates psoriatic skin: results of a randomised, double‐blind, placebo‐controlled phase Ib study . Exp Dermatol . 2023 ; 32 ( 9 ): 1538 - 45 .
Hua B , Yang M , Xue J , Dong C , Mao Y , Li O , et al . A novel single domain antibody targeting TIGIT for cancer use in combination therapies . Cancer Res . 2021 ; 81 ( Suppl 13 ): 2451 .
Deiteren A , Krupka E , Bontinck L , Imberdis K , Conickx G , Bas S , et al . A proof-of-mechanism trial in asthma with lunsekimig, a bispecific nanobody molecule . Eur Respir J . 2025 ; 65 ( 4 ): 2401461 .
An G , Xu Y , Yi S , Lyu R , Huang J , Li W , et al . A phase Ⅰb/Ⅱ study of trovocabtagene autoleucel (C-CAR088), an anti-BCMA CAR T-cell therapy in relapsed or refractory multiple myeloma . Blood . 2024 ; 144 ( Suppl 1 ): 7235 - 6 .
Mehra N , Voortman J , Wise DR , Calvo E , Piulats JM , Gonzalez-Billalabeitia E , et al . Phase Ⅰ/Ⅱ trial of LAVA-1207, a novel bispecific gamma-delta T-cell engager alone, or with low dose il-2 or pembrolizumab, in metastatic castration resistant prostate cancer (mcrpc) . J Clin Oncol . 2024 ; 42 ( 16 ): TPS2672 .
Jester BW , Zhao H , Gewe M , Adame T , Perruzza L , Bolick DT , et al . Development of spirulina for the manufacture and oral delivery of protein therapeutics . Nat Biotechnol . 2022 ; 40 ( 6 ): 956 - 64 .
D’Huyvetter M , Vos JD , Caveliers V , Vaneycken I , Heemskerk J , Duhoux FP , et al . Phase Ⅰ trial of 131 I-GMIB-anti-HER2-VHH1, a new promising candidate for HER2-targeted radionuclide therapy in breast cancer patients . J Nucl Med . 2021 ; 62 ( 8 ): 1097 - 105 .
Keller S , Kunz U , Schmid U , Beusmans J , Büchert M , He M , et al . Comprehensive biomarker and modeling approach to support dose finding for BI836880, a VEGF/ANG-2 inhibitor . J Transl Med . 2024 ; 22 ( 1 ): 934 .
Cunningham S , Piedra PA , Martinon-Torres F , Szymanski H , Brackeva B , Dombrecht E , et al . Nebulised ALX-0171 for respiratory syncytial virus lower respiratory tract infection in hospitalised children: a double-blind, randomised, placebo-controlled, phase 2b trial . Lancet Respir Med . 2021 ; 9 ( 1 ): 21 - 32 .
Nurbhai S , Roberts KJ , Carlton TM , Maggiore L , Cubitt MF , Ray KP , et al . Oral anti-tumour necrosis factor domain antibody V565 provides high intestinal concentrations, and reduces markers of inflammation in ulcerative colitis patients . Sci Rep . 2019 ; 9 ( 1 ): 14042 .
Haddley, K . Anti-interleukin-6 receptor subunit alpha (CD126; IL-6R), treatment of rheumatoid arthritis, treatment of systemic lupus erythematosus . Drugs Future . 2018 ; 43 ( 12 ): 891 - 900 .
Zhou Z , Han Y , Pan H , Sang C , Shi D , Feng C , et al . Tri-specific CD19xCD20xCD22 vhh CAR-T cells (LCAR-AIO) eradicate antigen-heterogeneous B cell tumors, enhance expansion, and prolong persistence in preclinical in vivo models . Blood . 2021 ; 138 ( Suppl 1 ): 1700 .
Mamdani H , Hecht JR , Sanborn RE , Palanca-Wessels MC , Yan M , Gutierrez M , et al . Abstract CT078: phase 1 study of SGN-EGFRD2 in solid tumors (SGNEGFRD2-001) . Cancer Res . 2024 ; 84 ( Suppl 7 ): CT078 .
Wan Y , Gai J , Zhu M , Li G , Qiao P . First-in-class inhalable anti-TSLP single domain antibody with therapeutic potential in asthma treatment . Am J Respir Crit Care Med . 2023 ; 207 : A1378 .
Hughes DJ , Chand G , Goh V , Cook GJR . Retraction note to: inter-and intraobserver agreement of the quantitative assessment of [ 99m Tc ] -labelled anti-programmed death-ligand 1 (PD-L1) spect/ct in non-small cell lung cancer . EJNMMI Res . 2021 ; 11 ( 1 ): 102 .
Wei Q . BioCon China 2021. Shanghai, China - April 22-23, 2021 . Drugs Future . 2021 ; 46 ( 7 ): 579 .
Bihlet AR , Balchen T , Goteti K , Sonne J , Ladel C , Karsdal MA , et al . Safety, tolerability, and pharmacodynamics of the ADAMTS-5 nanobody M6495: two phase 1, single‐center, double‐blind, randomized, placebo‐controlled studies in healthy subjects and patients with osteoarthritis . ACR Open Rheumatol . 2024 ; 6 ( 4 ): 205 - 13 .
Misra S , Kilari S , Yang B , Sharma A , Wu CC , Vazquez-Padron RI , et al . Anti human CX3CR1 vhh molecule attenuates venous neointimal hyperplasia of arteriovenous fistula in mouse model . J Am Soc Nephrol . 2021 ; 32 ( 7 ): 1630 - 48 .
Duggan S . Caplacizumab: first global approval . Drugs . 2018 ; 78 ( 15 ): 1639 - 42 .
Coppo P , Bubenheim M , Benhamou Y , Völker L , Brinkkötter P , Kühne L , et al . Caplacizumab use in immune-mediated thrombotic thrombocytopenic purpura: an international multicentre retrospective Cohort study (the Capla 1000 + project) . EClinicalMedicine . 2025 ; 82 : 103168 .
Al-Mashdali AF , Salih SM , Vatsyayan PA , Mudawi D , Elkourashy SA , Cherif H , et al . Caplacizumab for pediatric immune thrombotic thrombocytopenic purpura: a scoping review of current evidence . Blood Rev . 2025 ; 74 : 101326 .
Chen M , Jiang M , Wang X , Shen L , Li J . Envafolimab-first PD-1/PD-L1 antibody to be administered by subcutaneous injection for microsatellite instability-high or deficient mismatch repair advanced solid tumors . Expert Opin Biol Ther . 2022 ; 22 ( 10 ): 1227 - 32 .
Sidana S , Patel KK , Peres LC , Bansal R , Kocoglu MH , Shune L , et al . Safety and efficacy of standard-of-care ciltacabtagene autoleucel for relapsed/refractory multiple myeloma . Blood . 2025 ; 145 ( 1 ): 85 - 97 .
Jagannath S , Martin TG , Lin Y , Cohen AD , Raje N , Htut M , et al . Long-term (≥5-year) remission and survival after treatment with ciltacabtagene autoleucel in CARTITUDE-1 patients with relapsed/refractory multiple myeloma . J Clin Oncol . 2025 ; 43 ( 25 ): 2766 - 71 .
Martin T , Usmani SZ , Berdeja JG , Agha M , Cohen AD , Hari P , et al . Ciltacabtagene autoleucel, an anti-B-cell maturation antigen chimeric antigen receptor T-cell therapy, for relapsed/refractory multiple myeloma: CARTITUDE-12-year follow-up . J Clin Oncol . 2023 ; 41 ( 6 ): 1265 - 74 .
Tsumoto K , Takeuchi T . Next-generation anti-TNFα agents: the example of ozoralizumab . BioDrugs . 2024 ; 38 ( 3 ): 341 - 51 .
Watanabe R , Tanaka Y , Takeuchi T , Okamoto S , Kyuuma M , Matsumoto R , et al . Ozoralizumab shows effectiveness regardless of baseline RF and acpa titres in patients with RA: a post hoc analysis of the ohzora trial . Rheumatology . 2025 ; 64 ( 7 ): 4190 - 9 .
Verhaar ER , Woodham AW , Ploegh HL . Nanobodies in cancer . Semin Immunol . 2021 ; 52 : 101425 .
Roovers RC , Vosjan MJ , Laeremans T , el Khoulati R , de Bruin RC , Ferguson KM , et al . A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth . Int J Cancer . 2011 ; 129 ( 8 ): 2013 - 24 .
Maali A , Gholizadeh M , Feghhi-Najafabadi S , Noei A , Seyed-Motahari SS , Mansoori S , et al . Nanobodies in cell-mediated immunotherapy: on the road to fight cancer . Front Immunol . 2023 ; 14 : 1012841 .
Tydings CW , Singh B , Smith AW , Ledwitch KV , Brown BP , Lovly CM , et al . Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics . Protein Sci . 2024 ; 33 ( 10 ): e5141 .
Xu Z , Qiu C , Wen B , Wang S , Zhu L , Zhao L , et al . A bispecific nanobody targeting the dimerization interface of epidermal growth factor receptor: evidence for tumor suppressive actions in vitro and in vivo . Biochem Biophys Res Commun . 2021 ; 548 : 78 - 83 .
Schanzer JM , Wartha K , Moessner E , Hosse RJ , Moser S , Croasdale R , et al . XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1r and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer . MAbs . 2016 ; 8 ( 4 ): 811 - 27 .
Liu L , Tu B , Sun Y , Liao L , Lu X , Liu E , et al . Nanobody-based drug delivery systems for cancer therapy . J Control Release . 2025 ; 381 : 113562 .
Lutz EA , Jailkhani N , Momin N , Huang Y , Sheen A , Kang BH , et al . Intratumoral nanobody-IL-2 fusions that bind the tumor extracellular matrix suppress solid tumor growth in mice . PNAS Nexus . 2022 ; 1 ( 5 ): pgac244 .
Rahman MM , Wang J , Wang G , Su Z , Li Y , Chen Y , et al . Chimeric nanobody-decorated liposomes by self-assembly . Nat Nanotechnol . 2024 ; 19 ( 6 ): 818 - 24 .
Peruzzi JA , Vu TQ , Gunnels TF , Kamat NP . Rapid generation of therapeutic nanoparticles using cell‐free expression systems . Small Methods . 2023 ; 7 ( 12 ): e2201718 .
Kuno K , Shimane Y , Sakai Y , Kuruma Y . Cell-free production of target-directed liposomes utilizing membrane localization peptides . J Control Release . 2025 ; 388 ( Pt 1 ): 114343 .
Lu J , Jiang G . The journey of CAR-T therapy in hematological malignancies . Mol Cancer . 2022 ; 21 ( 1 ): 194 .
Lu P , Zhang X , Yang J , Li J , Qiu L , Gong M , et al . Nanobody-based naturally selected CD7-targeted CAR-T therapy for acute myeloid leukemia . Blood . 2025 ; 145 ( 10 ): 1022 - 33 .
Zeng Z , Roobrouck A , Deschamps G , Bonnevaux H , Guerif S , De Brabandere V , et al . Dual-targeting CD33/CD123 nanobody T-cell engager with potent anti-AML activity and good safety profile . Blood Adv . 2024 ; 8 ( 9 ): 2059 - 73 .
Ahmadi S , Knerr JM , Argemi L , Bordon KCF , Pucca MB , Cerni FA , et al . Scorpion venom: detriments and benefits . Biomedicines . 2020 ; 8 ( 5 ): 118 .
Machado Marinho AC , Chapeaurouge A , Dutra BM , Quintela BCSF , Pereira SS , Fernandes CFC . The role of venom proteomics and single-domain antibodies for antivenoms: progress in snake envenoming treatment . Drug Discov Today . 2024 ; 29 ( 5 ): 103967 .
Alirahimi E , Kazemi-Lomedasht F , Shahbazzadeh D , Habibi-Anbouhi M , Hosseininejad Chafi M , Sotoudeh N , et al . Nanobodies as novel therapeutic agents in envenomation . Biochim Biophys Acta Gen Subj . 2018 ; 1862 ( 12 ): 2955 - 65 .
Jenkins TP , Fryer T , Dehli RI , Jürgensen JA , Fuglsang-Madsen A , Føns S , et al . Toxin neutralization using alternative binding proteins . Toxins . 2019 ; 11 ( 1 ): 53 .
Bailon Calderon H , Yaniro Coronel VO , Cáceres Rey OA , Colque Alave EG , Leiva Duran WJ , Padilla Rojas C , et al . Development of nanobodies against hemorrhagic and myotoxic components of Bothrops atrox snake venom . Front Immunol . 2020 ; 11 : 655 .
D’Amélio F , Vigerelli H , de Brandão Prieto da Silva ÁR , Kerkis I . Bothrops moojeni venom and its components – an overview . J Venom Res . 2021 ; 11 : 26 - 33 .
Benard-Valle M , Wouters Y , Ljungars A , Nguyen GTT , Ahmadi S , Ebersole TW , et al . In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model . Nat Commun . 2024 ; 15 ( 1 ): 4310 .
Ahmadi S , Burlet NJ , Benard-Valle M , Guadarrama-Martínez A , Kerwin S , Cardoso IA , et al . Nanobody-based recombinant antivenom for cobra, mamba and rinkhals bites . Nature . 2025 ; 647 ( 8090 ): 716 - 725 .
Hmila I , Saerens D , Abderrazek RB , Vincke C , Abidi N , Benlasfar Z , et al . A bispecific nanobody to provide full protection against lethal scorpion envenoming . FASEB J . 2010 ; 24 ( 9 ): 3479 - 89 .
Ben Abderrazek R , Ksouri A , Idoudi F , Dhaouadi S , Hamdi E , Vincke C , et al . Neutralizing dromedary-derived nanobodies against boti-like toxin from the most hazardous scorpion venom in the middle east and North Africa region . Front Immunol . 2022 ; 13 : 863012 .
Rizk SS , Moustafa DM , ElBanna SA , Nour El-Din HT , Attia AS . Nanobodies in the fight against infectious diseases: repurposing nature’s tiny weapons . World J Microbiol Biotechnol . 2024 ; 40 ( 7 ): 209 .
Mei Y , Chen Y , Sivaccumar JP , An Z , Xia N , Luo W . Research progress and applications of nanobody in human infectious diseases . Front Pharmacol . 2022 ; 13 : 963978 .
Wilken L , McPherson A . Application of camelid heavy-chain variable domains (VHHs) in prevention and treatment of bacterial and viral infections . Int Rev Immunol . 2018 ; 37 ( 1 ): 69 - 76 .
Hanke L , Vidakovics Perez L , Sheward DJ , Das H , Schulte T , Moliner-Morro A , et al . An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction . Nat Commun . 2020 ; 11 ( 1 ): 4420 .
Huo J , Le Bas A , Ruza RR , Duyvesteyn HME , Mikolajek H , Malinauskas T , et al . Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2 . Nat Struct Mol Biol . 2020 ; 27 ( 9 ): 846 - 54 .
Xiang Y , Xu J , McGovern BL , Ranzenigo A , Huang W , Sang Z , et al . Adaptive multi-epitope targeting and avidity-enhanced nanobody platform for ultrapotent, durable antiviral therapy . Cell . 2024 ; 187 ( 24 ): 6966 - 6980.e23 .
Zhou T , Chen L , Gorman J , Wang S , Kwon YD , Lin BC , et al . Structural basis for llama nanobody recognition and neutralization of HIV-1 at the CD4-binding site . Structure . 2022 ; 30 ( 6 ): 862 - 875.e4 .
Zhu L , Huang B , Wang X , Ni F , Ao M , Wang R , et al . Highly potent and broadly neutralizing anti-CD4 trimeric nanobodies inhibit HIV-1 infection by inducing CD4 conformational alteration . Nat Commun . 2024 ; 15 ( 1 ): 6961 .
Xu J , Zhou T , McKee K , Zhang B , Liu C , Nazzari AF , et al . Ultrapotent broadly neutralizing human‐llama bispecific antibodies against HIV‐1 . Adv Sci . 2024 ; 11 ( 26 ): 2309268 .
Hansman GS , Kher G , Svirina AD , Tame JRH , Hartley-Tassell L , Irie H , et al . Development of a broad-spectrum therapeutic Fc-nanobody for human noroviruses . J Virol . 2024 ; 98 ( 7 ): e0070724 .
De Greve H , Fioravanti A . Single domain antibodies from camelids in the treatment of microbial infections . Front Immunol . 2024 ; 15 : 1334829 .
Jiang W , Huang C , Muyldermans S , Jia L . Small but mighty: nanobodies in the fight against infectious diseases . Biomolecules . 2025 ; 15 ( 5 ): 610 .
Bessalah S , Fairbrother JM , Salhi I , Vanier G , Khorchani T , Seddik MM , et al . Antimicrobial resistance and molecular characterization of virulence genes, phylogenetic groups of Escherichia coli isolated from diarrheic and healthy camel-calves in Tunisia . Comp Immunol Microbiol Infect Dis . 2016 ; 49 : 1 - 7 .
Salhi I , Bessalah S , Snoun D , Khorchani T , Hammadi M . Construction of a nanobodies phage display library from an Escherichia coli immunized dromedary . Iran J Biotechnol . 2020 ; 18 ( 1 ): e2247 .
Redrado‐Hernández S , Macías‐León J , Castro‐López J , Sanz AB , Dolader E , Arias M , et al . Broad protection against invasive fungal disease from a nanobody targeting the active site of fungal β‐1,3‐glucanosyltransferases . Angew Chem Int Ed Engl . 2024 ; 63 ( 34 ): e202405823 .
Ukegbu CV , Giorgalli M , Tapanelli S , Rona LDP , Jaye A , Wyer C , et al . PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector . Proc Natl Acad Sci U S A . 2020 ; 117 ( 13 ): 7363 - 73 .
Ukegbu CV , Mohamed M , Hoermann A , Qin Y , Kweyamba PA , Lwetoijera DW , et al . Nanobody-mediated targeting of Plasmodium falciparum PfPIMMS43 can block malaria transmission in mosquitoes . Commun Biol . 2025 ; 8 ( 1 ): 683 .
Bocancia-Mateescu LA , Stan D , Mirica AC , Ghita MG , Stan D , Ruta LL . Nanobodies as diagnostic and therapeutic tools for cardiovascular diseases (CVDs) . Pharmaceuticals . 2023 ; 16 ( 6 ): 863 .
Broggini L , Giono M , Speranzini V , Barzago M , Palladini G , Diomede L , et al . Nanobodies as novel potential drugs to target cardiac light chain amyloidosis . Cardiovasc Res . 2022 ; 118 : cvac066.233 .
McMahon C , Staus DP , Wingler LM , Wang J , Skiba MA , Elgeti M , et al . Synthetic nanobodies as angiotensin receptor blockers . Proc Natl Acad Sci U S A . 2020 ; 117 ( 33 ): 20284 - 91 .
Xu P , Zhang Y , Guo J , Li H , Konrath S , Zhou P , et al . A single-domain antibody targeting factor Ⅻ inhibits both thrombosis and inflammation . Nat Commun . 2024 ; 15 ( 1 ): 7898 .
Danis C , Dupré E , Zejneli O , Caillierez R , Arrial A , Bégard S , et al . Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment . Mol Ther . 2022 ; 30 ( 4 ): 1484 - 99 .
Monteiro AR , Barbosa DJ , Remião F , Silva R . Alzheimer’s disease: insights and new prospects in disease pathophysiology, biomarkers and disease-modifying drugs . Biochem Pharmacol . 2023 ; 211 : 115522 .
Benn J , Cheng S , Keeling S , Smith AE , Vaysburd MJ , Böken D , et al . Aggregate-selective removal of pathological tau by clustering-activated degraders . Science . 2024 ; 385 ( 6712 ): 1009 - 16 .
Zhao L , Meng F , Li Y , Liu S , Xu M , Chu F , et al . Multivalent nanobody conjugate with rigid, reactive oxygen species scavenging scaffold for multi‐target therapy of Alzheimer’s disease . Adv Mater . 2023 ; 35 ( 17 ): e2210879 .
Gu L , Li C , Lin J , Wang Q , Yin M , Zhang L , et al . Drug-loaded mesoporous carbon with sustained drug release capacity and enhanced antifungal activity to treat fungal keratitis . Biomater Adv . 2022 ; 136 : 212771 .
Liu X , Sui J , Li C , Wang Q , Peng X , Meng F , et al . The preparation and therapeutic effects of β-glucan-specific nanobodies and nanobody-natamycin conjugates in fungal keratitis . Acta Biomater . 2023 ; 169 : 398 - 409 .
Siebuhr AS , Werkmann D , Bay-Jensen AC , Thudium CS , Karsdal MA , Serruys B , et al . The anti-ADAMTS-5 nanobody ® M6495 protects cartilage degradation ex vivo . Int J Mol Sci . 2020 ; 21 ( 17 ): 5992 .
Zhu M , Ma L , Zhong P , Huang J , Gai J , Li G , et al . A novel inhalable nanobody targeting IL-4Rα for the treatment of asthma . J Allergy Clin Immunol . 2024 ; 154 ( 4 ): 1008 - 21 .
Tabatabaei MS , Ahmed M . Enzyme-linked immunosorbent assay (ELISA) . Methods Mol Biol . 2022 ; 2508 : 115 - 134 .
Zhu J , Liu Q , Li L , Zhang R , Chang Y , Zhao J , et al . Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies . Virol Sin . 2024 ; 39 ( 3 ): 478 - 89 .
Liao X , Wang J , Guo B , Bai M , Zhang Y , Yu G , et al . Enhancing nanobody immunoassays through ferritin fusion: construction of a Salmonella -specific fenobody for improved avidity and sensitivity . J Agric Food Chem . 2024 ; 72 ( 26 ): 14967 - 74 .
Koczula KM , Gallotta A . Lateral flow assays . Essays Biochem . 2016 ; 60 ( 1 ): 111 - 20 .
Pinto Torres JE , Goossens J , Ding J , Li Z , Lu S , Vertommen D , et al . Development of a nanobody-based lateral flow assay to detect active Trypanosoma congolense infections . Sci Rep . 2018 ; 8 ( 1 ): 9019 .
Xiao Y , Dong H , Wu C , Zhang K , Jiang X , Chen J , et al . Nanobody in a double “Y”-shaped assembly: a promising candidate for lateral flow immunoassays . Anal Chem . 2024 ; 96 ( 18 ): 7130 - 7 .
Katz HE . Antigen sensing via nanobody-coated transistors . Nat Biomed Eng . 2021 ; 5 ( 7 ): 639 - 40 .
El-Moghazy AY , Huo J , Amaly N , Vasylieva N , Hammock BD , Sun G . An innovative nanobody-based electrochemical immunosensor using decorated nylon nanofibers for point-of-care monitoring of human exposure to pyrethroid insecticides . ACS Appl Mater Interfaces . 2020 ; 12 ( 5 ): 6159 - 68 .
Yang Y , Inoue A , Yasuda T , Ueda H , Zhu B , Kitaguchi T . BRET nano Q-body: a nanobody-based ratiometric bioluminescent immunosensor for point-of-care testing . ACS Sens . 2024 ; 9 ( 11 ): 5955 - 65 .
Ma G , Holland CV , Wang T , Hofmann A , Fan CK , Maizels RM , et al . Human toxocariasis . Lancet Infect Dis . 2018 ; 18 ( 1 ): e14 - 24 .
Morales-Yánez F , Trashin S , Hermy M , Sariego I , Polman K , Muyldermans S , et al . Fast one-step ultrasensitive detection of Toxocara canis antigens by a nanobody-based electrochemical magnetosensor . Anal Chem . 2019 ; 91 ( 18 ): 11582 - 8 .
January J , Zwaenepoel O , Gettemans J , Iwuoha E . Highly sensitive nanobody-immunosensor for macrophage-capping protein . Biosens Bioelectron . 2025 ; 287 : 117681 .
Debie P , Lafont C , Defrise M , Hansen I , Van Willigen DM , Van Leeuwen FWB , et al . Size and affinity kinetics of nanobodies influence targeting and penetration of solid tumours . J Control Release . 2020 ; 317 : 34 - 42 .
Berland L , Kim L , Abousaway O , Mines A , Mishra S , Clark L , et al . Nanobodies for medical imaging: about ready for prime time? . Biomolecules . 2021 ; 11 ( 5 ): 637 .
Zeven K , Lauwers Y , De Mey L , Debacker JM , De Pauw T , De Groof TWM , et al . Advancements in nuclear imaging using radiolabeled nanobody tracers to support cancer immunotherapy . Immunother Adv . 2024 ; 4 ( 1 ): ltae006 .
Boekestijn I , van Oosterom MN , Dell’Oglio P , Van Velden FHP , Pool M , Maurer T , et al . The current status and future prospects for molecular imaging-guided precision surgery . Cancer Imaging . 2022 ; 22 ( 1 ): 48 .
Mateusiak Ł , Floru S , De Groof TWM , Wouters J , Declerck NB , Debie P , et al . Generation and characterization of novel pan-cancer anti-uPAR fluorescent nanobodies as tools for image-guided surgery . Adv Sci . 2024 ; 11 ( 30 ): 2400700 .
Sun Y , Hao Z , Gao H , Yang G , Pan B , Zhu M , et al . [99mTc] Tc-MY6349 probe for Trop2-targeted SPECT imaging: from preclinical to pilot clinical study . J Nucl Med . 2025 ; 66 ( 4 ): 543 - 51 .
Hernot S , Unnikrishnan S , Du Z , Shevchenko T , Cosyns B , Broisat A , et al . Nanobody-coupled microbubbles as novel molecular tracer . J Control Release . 2012 ; 158 ( 2 ): 346 - 53 .
Kaufmann BA , Sanders JM , Davis C , Xie A , Aldred P , Sarembock IJ , et al . Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1 . Circulation . 2007 ; 116 ( 3 ): 276 - 84 .
Punjabi M , Xu L , Ochoa-Espinosa A , Kosareva A , Wolff T , Murtaja A , et al . Ultrasound molecular imaging of atherosclerosis with nanobodies: translatable microbubble targeting murine and human VCAM (vascular cell adhesion molecule) 1 . Arterioscler Thromb Vasc Biol . 2019 ; 39 : 2520 - 30 .
Smith DA , Beaumont K , Maurer TS , Di L . Relevance of half-life in drug design . J Med Chem . 2018 ; 61 ( 10 ): 4273 - 82 .
Tayyab S , Feroz SR . Serum albumin: clinical significance of drug binding and development as drug delivery vehicle . Adv Protein Chem Struct Biol . 2021 ; 123 : 193 - 218 .
Tanaka Y . Ozoralizumab: first Nanobody ® therapeutic for rheumatoid arthritis . Expert Opin Biol Ther . 2023 ; 23 ( 7 ): 579 - 87 .
Li Q , Kong Y , Zhong Y , Huang A , Ying T , Wu Y . Half‐life extension of single‐domain antibody-drug conjugates by albumin binding moiety enhances antitumor efficacy . MedComm . 2024 ; 5 ( 5 ): e557 .
Cheng Y , Hao Y , Bao F , Zhang H , Liu Y , Ao K , et al . Preparation and identification of a single domain antibody specific for adenovirus vectors and its application to the immunoaffinity purification of adenoviruses . AMB Express . 2022 ; 12 ( 1 ): 80 .
Verheesen P , ten Haaft MR , Lindner N , Verrips CT , De Haard JJW . Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immunoperfusion chromatography . Biochim Biophys Acta . 2003 ; 1624 ( 1-3 ): 21 - 8 .
Cong ATQ , Witter TL , Schellenberg MJ . High‐efficiency recombinant protein purification using mCherry and YFP nanobody affinity matrices . Protein Sci . 2022 ; 31 ( 9 ): e4383 .
Stevens TA , Tomaleri GP , Hazu M , Wei S , Nguyen VN , DeKalb C , et al . A nanobody-based strategy for rapid and scalable purification of human protein complexes . Nat Protoc . 2024 ; 19 ( 1 ): 127 - 58 .
Prozzillo Y , Fattorini G , Santopietro MV , Suglia L , Ruggiero A , Ferreri D , et al . Targeted protein degradation tools: overview and future perspectives . Biology . 2020 ; 9 ( 12 ): 421 .
Ibrahim AFM , Shen L , Tatham MH , Dickerson D , Prescott AR , Abidi N , et al . Antibody RING-mediated destruction of endogenous proteins . Mol Cell . 2020 ; 79 ( 1 ): 155 - 166.e9 .
Jiang S , Lv X , Ouyang Z , Chi H , Zeng Y , Wang Y , et al . Programmable circular multivalent nanobody‐targeting chimeras (mNbTACs) for multireceptor‐mediated protein degradation and targeted drug delivery . Angew Chem Int Ed Engl . 2024 ; 63 ( 52 ): ee202407986 .
Zhang H , Han Y , Yang Y , Lin F , Li K , Kong L , et al . Covalently engineered nanobody chimeras for targeted membrane protein degradation . J Am Chem Soc . 2021 ; 143 ( 40 ): 16377 - 82 .
Liu H , Fu Z , Han Y , Fang Y , Shen W , Chen Z , et al . Conditionally activatable chimeras for tumor-specific membrane protein degradation . J Am Chem Soc . 2024 ; 146 ( 48 ): 32933 - 41 .
García-Nafría J , Tate CG . Structure determination of GPCRS: cryo-EM compared with X-ray crystallography . Biochem Soc Trans . 2021 ; 49 ( 5 ): 2345 - 55 .
Koide S . Engineering of recombinant crystallization chaperones . Curr Opin Struct Biol . 2009 ; 19 ( 4 ): 449 - 57 .
Robertson MJ , Papasergi-Scott MM , He F , Seven AB , Meyerowitz JG , Panova O , et al . Structure determination of inactive-state GPCRS with a universal nanobody . Nat Struct Mol Biol . 2022 ; 29 ( 12 ): 1188 - 95 .
Uchański T , Masiulis S , Fischer B , Kalichuk V , López-Sánchez U , Zarkadas E , et al . Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM . Nat Methods . 2021 ; 18 ( 1 ): 60 - 8 .
Bloch JS , Mukherjee S , Kowal J , Filippova EV , Niederer M , Pardon E , et al . Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins . Proc Natl Acad Sci U S A . 2021 ; 118 ( 47 ): e2115435118 .
Muyldermans S . Applications of nanobodies . Annu Rev Anim Biosci . 2021 ; 9 : 401 - 21 .
Wagner TR , Rothbauer U . Nanobodies right in the middle: intrabodies as toolbox to visualize and modulate antigens in the living cell . Biomolecules . 2020 ; 10 ( 12 ): 1701 .
Schmidthals K , Helma J , Zolghadr K , Rothbauer U , Leonhardt H . Novel antibody derivatives for proteome and high-content analysis . Anal Bioanal Chem . 2010 ; 397 ( 8 ): 3203 - 8 .
Kuo CW , Gök C , Fulton H , Dickson-Murray E , Adu S , Gallen EK , et al . Nanobody-thioesterase chimeras to specifically target protein palmitoylation . Nat Commun . 2025 ; 16 ( 1 ): 1445 .
Li Q , Liu H , Du X , Xie Y , Chen Y , Qiu J , et al . Na nobody-assisted nanoluciferase fragment complementation for in situ measurement and visualization of endogenous protein-protein interaction . Biosens Bioelectron . 2025 ; 272 : 117102 .
Stuart T , Hao S , Zhang B , Mekerishvili L , Landau DA , Maniatis S , et al . Nanobody-tethered transposition enables multifactorial chromatin profiling at single-cell resolution . Nat Biotechnol . 2023 ; 41 ( 6 ): 806 - 12 .
Kalogriopoulos NA , Tei R , Yan Y , Klein PM , Ravalin M , Cai B , et al . Synthetic GPCRS for programmable sensing and control of cell behaviour . Nature . 2025 ; 637 ( 8044 ): 230 - 9 .
Zhang Y , Chen Z , Wang X , Yan R , Bao H , Chu X , et al . Site-specific tethering nanobodies on recombinant adeno-associated virus vectors for retargeted gene therapy . Acta Biomater . 2024 ; 187 : 304 - 15 .
Kibria MG , Akazawa-Ogawa Y , Rahman N , Hagihara Y , Kuroda Y . The immunogenicity of an anti-EGFR single domain antibody (VHH) is enhanced by misfolded amorphous aggregation but not by heat-induced aggregation . Eur J Pharm Biopharm . 2020 ; 152 : 164 - 74 .
Papadopoulos KP , Isaacs R , Bilic S , Kentsch K , Huet HA , Hofmann M , et al . Unexpected hepatotoxicity in a phase I study of TAS266, a novel tetravalent agonistic Nanobody ® targeting the DR5 receptor . Cancer Chemother Pharmacol . 2015 ; 75 ( 5 ): 887 - 95 .
Svecova D , Lubell MW , Casset-Semanaz F , Mackenzie H , Grenningloh R , Krueger JG . A randomized, double-blind, placebo-controlled phase 1 study of multiple ascending doses of subcutaneous M1095, an anti-interleukin 17A/F nanobody, in moderate-to-severe psoriasis . J Am Acad Dermatol . 2019 ; 81 ( 1 ): 196 - 203 .
Haworth R , Boyle M , Edwards P , Gupta R , Fagg R , Karantabias G , et al . Preclinical safety assessment of 2 inhaled single-domain antibodies in the cynomolgus monkey . Toxicol Pathol . 2021 ; 49 ( 2 ): 296 - 314 .
Harthorn A , Kuo TH , Torres SW , Lobb RR , Hackel BJ . Expression-dependent tumor pretargeting via engineered avidity . Mol Pharm . 2025 ; 22 ( 1 ): 558 - 72 .
Pejenaute A , Gálvez-Larrosa L , Segovia CM , Herrero-Alfonso P , Fernández-Ramos D , Lopitz-Otsoa F , et al . Improving the production and stability of nanobodies . Protein Eng Des Sel . 2025 ; 38 : gzaf010 .
Ma L , Zhu M , Gai J , Li G , Chang Q , Qiao P , et al . Preclinical development of a novel CD47 nanobody with less toxicity and enhanced anti-cancer therapeutic potential . J Nanobiotechnology . 2020 ; 18 ( 1 ): 12 .
Li X , Qiao P , Zhang Y , Liu G , Zhu M , Gai J , et al . High performance production process development and scale-up of an anti-TSLP nanobody . Protein Expr Purif . 2024 ; 218 : 106441 .
Kamoshida G , Yamaguchi D , Kaya Y , Yamakado T , Yamashita K , Aoyagi M , et al . Development of a novel bacterial production system for recombinant bioactive proteins completely free from endotoxin contamination . PNAS Nexus . 2024 ; 3 : pgae328 .
Zhu H , Ding Y . Nanobodies: from discovery to AI-driven design . Biology . 2025 ; 14 ( 5 ): 547 .
Liu J , Wu L , Xie A , Liu W , He Z , Wan Y , et al . Unveiling the new chapter in nanobody engineering: advances in traditional construction and AI-driven optimization . J Nanobiotechnology . 2025 ; 23 ( 1 ): 87 .
Abramson J , Adler J , Dunger J , Evans R , Green T , Pritzel A , et al . Accurate structure prediction of biomolecular interactions with AlphaFold 3 . Nature . 2024 ; 630 ( 8016 ): 493 - 500 .
Krishna R , Wang J , Ahern W , Sturmfels P , Venkatesh P , Kalvet I , et al . Generalized biomolecular modeling and design with RoseTTAFold All-Atom . Science . 2024 ; 384 ( 6693 ): eadl2528 .
Kortemme T . De novo protein design — from new structures to programmable functions . Cell . 2024 ; 187 ( 3 ): 526 - 44 .
Lauko A , Pellock SJ , Sumida KH , Anishchenko I , Juergens D , Ahern W , et al . Computational design of serine hydrolases . Science . 2025 ; 388 ( 6744 ): eadu2454 .
Liu H , Song Z , Zhang Y , Wu B , Chen D , Zhou Z , et al . De novo design of self-assembling peptides with antimicrobial activity guided by deep learning . Nat Mater . 2025 ; 24 ( 8 ): 1295 - 306 .
Koh HY , Zheng Y , Yang M , Arora R , Webb GI , Pan S , et al . AI-driven protein design . Nat Rev Bioeng . 2025 ; 3 : 1034 - 56 .
Watson JL , Juergens D , Bennett NR , Trippe BL , Yim J , Eisenach HE , et al . De novo design of protein structure and function with RFdiffusion . Nature . 2023 ; 620 ( 7976 ): 1089 - 100 .
Ahern W , Yim J , Tischer D , Salike S , Woodbury SM , Kim D , et al . Atom-level enzyme active site scaffolding using RFdiffusion2 . Nat Methods . 2026 ; 23 ( 1 ): 96 - 105 .
Butcher J , Krishna R , Mitra R , Brent RI , Li Y , Corley N , et al . De novo design of all-atom biomolecular interactions with RFdiffusion3 . bioRxiv [Preprint] . 2025 . doi: 10.1101/2025.09.18.676967 https://doi.org/10.1101/2025.09.18.676967 .
Dauparas J , Anishchenko I , Bennett N , Bai H , Ragotte RJ , Milles LF , et al . Robust deep learning-based protein sequence design using ProteinMPNN . Science . 2022 ; 378 ( 6615 ): 49 - 56 .
Zambaldi V , La D , Chu AE , Patani H , Danson AE , Kwan TOC , et al . De novo design of high-affinity protein binders with AlphaProteo . https://arxiv.org/abs/2409.08022 https://arxiv.org/abs/2409.08022 ( 2024 ). Accessed 26 Jan, 2026 .
Pacesa M , Nickel L , Schellhaas C , Schmidt J , Pyatova E , Kissling L , et al . One-shot design of functional protein binders with BindCraft . Nature . 2025 ; 646 ( 8084 ): 483 - 92 .
Zhang O , Zhang X , Lin H , Tan C , Wang Q , Mo Y , et al . ODesign: a world model for biomolecular interaction design . https://arxiv.org/abs/2510.22304 https://arxiv.org/abs/2510.22304 ( 2025 ). Accessed 26 Jan, 2026 .
Cho Y , Pacesa M , Zhang Z , Correia BE , Ovchinnikov S . Boltzdesign1: inverting all-atom structure prediction model for generalized biomolecular binder design . https://www.biorxiv.org/content/10.1101/2025.04.06.647261v1 https://www.biorxiv.org/content/10.1101/2025.04.06.647261v1 ( 2025 ). Accessed 26 Jan, 2026 .
Chai Discovery Team , Boitreaud J , Dent J , Geisz D , McPartlon M , Meier J , et al . Zero-shot antibody design in a 24-well plate . https://www.biorxiv.org/content/10.1101/2025.07.05.663018v1 https://www.biorxiv.org/content/10.1101/2025.07.05.663018v1 ( 2025 ). Accessed 26 Jan, 2026 .
Wang R , Wu F , Gao X , Wu J , Zhao P , Yao J . IgGM: a generative model for functional antibody and nanobody design . https://www.biorxiv.org/content/10.1101/2024.09.19.613838v2 https://www.biorxiv.org/content/10.1101/2024.09.19.613838v2 ( 2025 ). Accessed 26 Jan, 2026 .
Stark H , Faltings F , Choi M , Xie Y , Hur E , O’Donnell T , et al . BoltzGen: toward universal binder design . https://www.biorxiv.org/content/biorxiv/early/2025/11/24/2025.11.20.689494.full.pdf https://www.biorxiv.org/content/biorxiv/early/2025/11/24/2025.11.20.689494.full.pdf ( 2025 ). Accessed 26 Jan, 2026 .
Nabla Bio , Biswas S . De novo design of epitope-specific antibodies against soluble and multipass membrane proteins with high specificity, developability, and function . https://www.biorxiv.org/content/10.1101/2025.01.21.633066v1 https://www.biorxiv.org/content/10.1101/2025.01.21.633066v1 ( 2025 ). Accessed 26 Jan, 2026 .
Swanson E , Nichols M , Ravichandran S , Ogden P . mBER: controllable de novo antibody design with million-scale experimental screening . https://www.biorxiv.org/content/10.1101/2025.09.26.678877v1 https://www.biorxiv.org/content/10.1101/2025.09.26.678877v1 ( 2025 ). Accessed 26 Jan, 2026 .
Mille-Fragoso LS , Wang JN , Driscoll CL , Dai H , Widatalla T , Zhang X , et al . Efficient generation of epitope-targeted de novo antibodies with germinal . https://www.biorxiv.org/content/10.1101/2025.09.19.677421v2 https://www.biorxiv.org/content/10.1101/2025.09.19.677421v2 ( 2025 ). Accessed 26 Jan, 2026 .
Høie MH , Hummer AM , Olsen TH , Aguilar-Sanjuan B , Nielsen M , Deane CM . AntiFold: improved structure-based antibody design using inverse folding . Bioinform Adv . 2025 ; 5 ( 1 ): vbae202 .
Sun Z , Yuan J , Jaiswal D , Ge J , Liang T , Wei J , et al . AntiBMPNN: structure‐guided graph neural networks for precision antibody engineering . Adv Sci . 2025 ; 12 ( 35 ): e04278 .
Zhao S , Luo J , Xu P , Zeng J , Yan G , Yu F , et al . Designed peptide binders and nanobodies as protac starting points for targeted degradation of pcna and BCL6 . Int J Biol Macromol . 2025 ; 308 ( Pt 3 ): 142667 .
Bennett NR , Watson JL , Ragotte RJ , Borst AJ , See DL , Weidle C , et al . Atomically accurate de novo design of antibodies with RFdiffusion . Nature . 2026 ; 649 ( 8095 ): 183 - 93 .
Wang X , Chen H , Chen B , Liang L , Mei F , Huang B . NanoAbLLaMA: construction of nanobody libraries with protein large language models . Front Chem . 2025 ; 13 : 1545136 .
Xiong D , Ming Y , Li Y , Li S , Chen K , Liu J , et al . EvoNB: a protein language model-based workflow for nanobody mutation prediction and optimization . J Pharm Anal . 2025 ; 15 ( 6 ): 101260 .
He X , Li J , Xu J , Shan H , Shen S , Gao S , et al . AI-driven antibody design with generative diffusion models: current insights and future directions . Acta Pharmacol Sin . 2025 ; 46 ( 3 ): 565 - 74 .
Nguyen MN , Krutz NL , Limviphuvadh V , Lopata AL , Gerberick GF , Maurer-Stroh S . AllerCatPro 2.0: a web server for predicting protein allergenicity potential . Nucleic Acids Res . 2022 ; 50 ( W1 ): W36 - 43 .
Sharma N , Naorem LD , Jain S , Raghava GPS . ToxinPred2: an improved method for predicting toxicity of proteins . Brief Bioinform . 2022 ; 23 ( 5 ): bbac174 .
Blaabjerg LM , Kassem MM , Good LL , Jonsson N , Cagiada M , Johansson KE , et al . Rapid protein stability prediction using deep learning representations . ELife . 2023 ; 12 : e82593 .
Miotto M , Armaos A , Di Rienzo L , Ruocco G , Milanetti E , Tartaglia GG . Thermometer: a webserver to predict protein thermal stability . Bioinformatics . 2022 ; 38 ( 7 ): 2060 - 1 .
Eshak F , Goupil-Lamy A . Advancements in nanobody epitope prediction: a comparative study of AlphaFold2Multimer vs . AlphaFold3. J Chem Inf Model . 2025 ; 65 ( 4 ): 1782 - 97 .
Hitawala FN , Gray JJ . What does AlphaFold3 learn about antigen and nanobody docking, and what remains unsolved? . MAbs . 2025 ; 17 ( 1 ): 2545601 .
Xu S , Feng Q , Qiao L , Wu H , Shen T , Cheng Y , et al . Benchmarking all-atom biomolecular structure prediction with FoldBench . Nat Commun . 2025 ; 17 ( 1 ): 442 .
Maksymova L , Pilger YA , Nuhn L , Van Ginderachter JA . Nanobodies targeting the tumor microenvironment and their formulation as nanomedicines . Mol Cancer . 2025 ; 24 ( 1 ): 65 .
Feng Z , Zhang X , Peng Z , Aghamajidi A , Wu Y , Hua X . Nanobody-directed CEA-targeting CAR T cells eliminate gastrointestinal cancer xenografts . Cancer Immunol Res . 2025 ; 13 ( 8 ): 160 - 71 .
Feng Y , Meshaw R , McDougald D , Zhou Z , Zhao XG , Jannetti SA , et al . Evaluation of an 131 I-labeled HER2-specific single domain antibody fragment for the radiopharmaceutical therapy of HER2-expressing cancers . Sci Rep . 2022 ; 12 ( 1 ): 3020 .
Tripathy RK , Pande AH . Nanobody-oligonucleotide conjugates (NucleoBodies): the next frontier in oligonucleotide therapy . Pharm Res . 2025 ; 42 ( 2 ): 219 - 36 .
Boonkaew S , Szot-Karpińska K , Niedziółka-Jönsson J , De Marco A , Jönsson-Niedziółka M . NFC smartphone-based electrochemical microfluidic device integrated with nanobody recognition for C-reactive protein . ACS Sens . 2024 ; 9 ( 6 ): 3066 - 74 .
Qin S , Liu Y , He G , Yang J , Zeng F , Lu Q , et al . Spatiotemporal delivery of dual nanobodies by engineered probiotics to reverse tumor immunosuppression via targeting tumor-derived exosomes . ACS Nano . 2024 ; 18 ( 39 ): 26858 - 71 .
Abad C , Demeules M , Guillou C , Gondé H , Zoubairi R , Tan YV , et al . Administration of an AAV vector coding for a P2X7-blocking nanobody-based biologic ameliorates colitis in mice . J Nanobiotechnol . 2024 ; 22 ( 1 ): 27 .
Goebeler ME , Stuhler G , Bargou R . Bispecific and multispecific antibodies in oncology: opportunities and challenges . Nat Rev Clin Oncol . 2024 ; 21 ( 7 ): 539 - 60 .
Zhao Y , Ma Y , Zang A , Cheng Y , Zhang Y , Wang X , et al . First-in-human phase Ⅰ/Ⅰb study of QL1706 (PSB205), a bifunctional PD1/CTLA4 dual blocker, in patients with advanced solid tumors . J Hematol Oncol . 2023 ; 16 ( 1 ): 50 .
The trial reading is over. You can log in to your account, go to the personal center, purchase VIP membership, and read the full text.
Already a VIP member?
Log in >FULL TEXT LINK
0
Views
0
Downloads
0
CSCD
Alert me when the article has been cited
文章被引用时，请邮件提醒。
Submit
Tools
Download
- L-PDFDownload
- Meta-XMLDownload
Export Citation
- BibTeXDownload
- EndnoteDownload
- RefMan Download
- NoteExpressDownload
- NoteFirstDownload
Share
Share with wechat friends or circle of friends
Add to favorites
Add to my album
Publicity Resources
Related Articles
No data
Related Author
No data
Related Institution
No data
Switch Language
- Technical support is provided by Beijing Founder electronics co., LTD 京ICP备09064830号-19
京公网安备11010802024621 - It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
- Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.
0
Bulk Citation
Export to
BibTeXEndnoteRefManNoteExpressNoteFirst
TOP