Nanobodies targeting immune checkpoint molecules for tumor immunotherapy and immunoimaging (Review)

Yu,Sheng; Xiong,Gui; Zhao,Shimei; Tang,Yanbo; Tang,Hua; Wang,Kaili; Liu,Hongjing; Lan,Ke; Bi,Xiongjie; Duan,Siliang

doi:10.3892/ijmm.2020.4817

Nanobodies targeting immune checkpoint molecules for tumor immunotherapy and immunoimaging (Review)

Authors:
- Sheng Yu
- Gui Xiong
- Shimei Zhao
- Yanbo Tang
- Hua Tang
- Kaili Wang
- Hongjing Liu
- Ke Lan
- Xiongjie Bi
- Siliang Duan
View Affiliations

Affiliations: Department of Medicine, Guangxi University of Science and Technology, Liuzhou, Guangxi Zhuang Autonomous Region 545005, P.R. China, Department of Gastroenterology, The First Afﬁliated Hospital of Guangxi University of Science and Technology, Liuzhou, Guangxi Zhuang Autonomous Region 545001, P.R. China, Department of Clinical Laboratory, The Second Clinical Medical College of Guangxi University of Science and Technology, Liuzhou, Guangxi Zhuang Autonomous Region 545006, P.R. China, Department of Clinical Laboratory, The First Afﬁliated Hospital of Guangxi University of Science and Technology, Liuzhou, Guangxi Zhuang Autonomous Region 545001, P.R. China
Published online on: December 14, 2020 https://doi.org/10.3892/ijmm.2020.4817
Pages: 444-454
Copyright: © Yu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The immune checkpoint blockade is an effective strategy to enhance the anti‑tumor T cell effector activity, thus becoming one of the most promising immunotherapeutic strategies in the history of cancer treatment. Several immune checkpoint inhibitor have been approved by the FDA, such as anti‑CTLA‑4, anti‑PD‑1, anti‑PD‑L1 monoclonal antibodies. Most tumor patients benefitted from these antibodies, but some of the patients did not respond to them. To increase the effectiveness of immunotherapy, including immune checkpoint blockade therapies, miniaturization of antibodies has been introduced. A single‑domain antibody, also known as nanobody, is an attractive reagent for immunotherapy and immunoimaging thanks to its unique structural characteristic consisting of a variable region of a single heavy chain antibody. This structure confers to the nanobody a light molecular weight, making it smaller than conventional antibodies, although remaining able to bind to a specific antigen. Therefore, this review summarizes the production of nanobodies targeting immune checkpoint molecules and the application of nanobodies targeting immune checkpoint molecules in immunotherapy and immunoimaging.

View Figures

View References

1	Frankel T, Lanfranca MP and Zou W: The role of tumor micro-environment in cancer immunotherapy. Adv Exp Med Biol. 1036:51–64. 2017. View Article : Google Scholar
2	Fan CA, Reader J and Roque DM: Review of immune therapies targeting ovarian cancer. Curr Treat Options Oncol. 19:742018. View Article : Google Scholar : PubMed/NCBI
3	Marin-Acevedo JA, Soyano AE, Dholaria B, Knutson KL and Lou Y: Cancer immunotherapy beyond immune checkpoint inhibitors. J Hematol Oncol. 11:82018. View Article : Google Scholar : PubMed/NCBI
4	Marin-Acevedo JA, Dholaria B, Soyano AE, Knutson KL, Chumsri S and Lou Y: Next generation of immune checkpoint therapy in cancer: New developments and challenges. J Hematol Oncol. 11:392018. View Article : Google Scholar : PubMed/NCBI
5	Ok CY and Young KH: Checkpoint inhibitors in hematological malignancies. J Hematol Oncol. 10:1032017. View Article : Google Scholar : PubMed/NCBI
6	Baghdadi M, Takeuchi S, Wada H and Seino K: Blocking mono-clonal antibodies of TIM proteins as orchestrators of anti-tumor immune response. MAbs. 6:1124–1132. 2014. View Article : Google Scholar
7	Rodallec A, Sicard G, Fanciullino R, Benzekry S, Lacarelle B, Milano G and Ciccolini J: Turning cold tumors into hot tumors: Harnessing the potential of tumor immunity using nanoparticles. Expert Opin Drug Metab Toxicol. 14:1139–1147. 2018.PubMed/NCBI
8	Nishino M, Ramaiya NH, Hatabu H and Hodi FS: Monitoring immune-checkpoint blockade: Response evaluation and biomarker development. Nat Rev Clin Oncol. 14:655–668. 2017. View Article : Google Scholar : PubMed/NCBI
9	Darvin P, Toor SM, Sasidharan Nair V and Elkord E: Immune checkpoint inhibitors: Recent progress and potential biomarkers. Exp Mol Med. 50:1–11. 2018. View Article : Google Scholar : PubMed/NCBI
10	Hargadon KM, Johnson CE and Williams CJ: Immune checkpoint blockade therapy for cancer: An overview of FDA-approved immune checkpoint inhibitors. Int Immunopharmacol. 62:29–39. 2018. View Article : Google Scholar : PubMed/NCBI
11	Shen H, Yang ES, Conry M, Fiveash J, Contreras C, Bonner JA and Shi LZ: Predictive biomarkers for immune checkpoint blockade and opportunities for combination therapies. Genes Dis. 6:232–246. 2019. View Article : Google Scholar
12	Kamath SD, Kalyan A and Benson AB III: Pembrolizumab for the treatment of gastric cancer. Expert Rev Anticancer Ther. 18:1177–1187. 2018. View Article : Google Scholar : PubMed/NCBI
13	Hsu FS, Su CH and Huang KH: A comprehensive review of US FDA-approved immune checkpoint inhibitors in urothelial carcinoma. J Immunol Res. 2017:69405462017. View Article : Google Scholar
14	Song MK, Park BB and Uhm J: Understanding immune evasion and therapeutic targeting associated with PD-1/PD-L1 pathway in diffuse large B-cell lymphoma. Int J Mol Sci. 20:13262019. View Article : Google Scholar :
15	Michot JM, Bigenwald C, Champiat S, Collins M, Carbonnel F, Postel-Vinay S, Berdelou A, Varga A, Bahleda R, Hollebecque A, et al: Immune-related adverse events with immune checkpoint blockade: A comprehensive review. Eur J Cancer. 54:139–148. 2016. View Article : Google Scholar : PubMed/NCBI
16	Bannas P, Hambach J and Koch-Nolte F: Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Front Immunol. 8:16032017. View Article : Google Scholar : PubMed/NCBI
17	Ubah OC, Buschhaus MJ, Ferguson L, Kovaleva M, Steven J, Porter AJ and Barelle CJ: Next-generation flexible formats of VNAR domains expand the drug platform's utility and developability. Biochem Soc Trans. 46:1559–1565. 2018. View Article : Google Scholar : PubMed/NCBI
18	Wang H, Meng AM, Li SH and Zhou XL: A nanobody targeting carcinoembryonic antigen as a promising molecular probe for non-small cell lung cancer. Mol Med Rep. 16:625–630. 2017. View Article : Google Scholar : PubMed/NCBI
19	Broos K, Lecocq Q, Raes G, Devoogdt N, Keyaerts M and Breckpot K: Noninvasive imaging of the PD-1:PD-L1 immune checkpoint: Embracing nuclear medicine for the benefit of personalized immunotherapy. Theranostics. 8:3559–3570. 2018. View Article : Google Scholar : PubMed/NCBI
20	Mayer AT, Natarajan A, Gordon SR, Maute RL, McCracken MN, Ring AM, Weissman IL and Gambhir SS: Practical immuno-PET radiotracer design considerations for human immune checkpoint imaging. J Nucl Med. 58:538–546. 2017. View Article : Google Scholar :
21	Natarajan A, Mayer AT, Reeves RE, Nagamine CM and Gambhir SS: Development of novel ImmunoPET tracers to image human PD-1 checkpoint expression on tumor-infiltrating lymphocytes in a humanized mouse model. Mol Imaging Biol. 19:903–914. 2017. View Article : Google Scholar : PubMed/NCBI
22	Li M, Ehlerding EB, Jiang D, Barnhart TE, Chen W, Cao T, Engle JW and Cai W: In vivo characterization of PD-L1 expression in breast cancer by immuno-PET with ⁸⁹Zr-labeled avelumab. Am J Transl Res. 12:1862–1872. 2020.
23	Kikuchi M, Clump DA, Srivastava RM, Sun L, Zeng D, Diaz-Perez JA, Anderson CJ, Edwards WB and Ferris RL: Preclinical immunoPET/CT imaging using Zr-89-labeled anti-PD-L1 monoclonal antibody for assessing radiation-induced PD-L1 upregulation in head and neck cancer and melanoma. Oncoimmunology. 6:e13290712017. View Article : Google Scholar : PubMed/NCBI
24	Li D, Zou S, Cheng S, Song S, Wang P and Zhu X: Monitoring the response of PD-L1 expression to epidermal growth factor receptor tyrosine kinase inhibitors in nonsmall-cell lung cancer xenografts by immuno-PET imaging. Mol Pharm. 16:3469–3476. 2019. View Article : Google Scholar : PubMed/NCBI
25	González Trotter DE, Meng X, McQuade P, Rubins D, Klimas M, Zeng Z, Connolly BM, Miller PJ, O'Malley SS, Lin SA, et al: In vivo imaging of the programmed death ligand 1 by 18F PET. J Nucl Med. 58:1852–1857. 2017. View Article : Google Scholar
26	Hettich M, Braun F, Bartholomä MD, Schirmbeck R and Niedermann G: High-resolution PET imaging with therapeutic antibody-based PD-1/PD-L1 checkpoint tracers. Theranostics. 6:1629–1640. 2016. View Article : Google Scholar : PubMed/NCBI
27	Heskamp S, Hobo W, Molkenboer-Kuenen JD, Olive D, Oyen WJ, Dolstra H and Boerman OC: Noninvasive imaging of tumor PD-L1 expression using radiolabeled anti-PD-L1 antibodies. Cancer Res. 75:2928–2936. 2015. View Article : Google Scholar : PubMed/NCBI
28	Li D, Cheng S, Zou S, Zhu D, Zhu T, Wang P and Zhu X: Immuno-PET imaging of ⁸⁹Zr labeled anti-PD-L1 domain anti-body. Mol Pharm. 15:1674–1681. 2018. View Article : Google Scholar : PubMed/NCBI
29	Josefsson A, Nedrow JR, Park S, Banerjee SR, Rittenbach A, Jammes F, Tsui B and Sgouros G: Imaging, biodistribution, and dosimetry of radionuclide-labeled PD-L1 antibody in an immunocompetent mouse model of breast cancer. Cancer Res. 76:472–479. 2016. View Article : Google Scholar :
30	Natarajan A, Patel CB, Habte F and Gambhir SS: Dosimetry prediction for clinical translation of ⁶⁴Cu-pembrolizumab ImmunoPET targeting human PD-1 expression. Sci Rep. 8:6332018. View Article : Google Scholar
31	Van Audenhove I and Gettemans J: Nanobodies as versatile tools to understand, diagnose, visualize and treat cancer. EBioMedicine. 8:40–48. 2016. View Article : Google Scholar : PubMed/NCBI
32	Lecocq Q, De Vlaeminck Y, Hanssens H, D'Huyvetter M, Raes G, Goyvaerts C, Keyaerts M, Devoogdt N and Breckpot K: Theranostics in immunooncology using nanobody derivatives. Theranostics. 9:7772–7791. 2019. View Article : Google Scholar :
33	Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N and Hamers R: Naturally occurring antibodies devoid of light chains. Nature. 363:446–448. 1993. View Article : Google Scholar : PubMed/NCBI
34	Muyldermans S: Nanobodies: Natural single-domain antibodies. Annu Rev Biochem. 82:775–797. 2013. View Article : Google Scholar : PubMed/NCBI
35	Könning D, Zielonka S, Grzeschik J, Empting M, Valldorf B, Krah S, Schröter C, Sellmann C, Hock B and Kolmar H: Camelid and shark single domain antibodies: Structural features and therapeutic potential. Curr Opin Struct Biol. 45:10–16. 2017. View Article : Google Scholar
36	Krah S, Schröter C, Zielonka S, Empting M, Valldorf B and Kolmar H: Single-domain antibodies for biomedical applications. Immunopharmacol Immunotoxicol. 38:21–28. 2016. View Article : Google Scholar
37	Steeland S, Vandenbroucke RE and Libert C: Nanobodies as therapeutics: Big opportunities for small antibodies. Drug Discov Today. 21:1076–1113. 2016. View Article : Google Scholar : PubMed/NCBI
38	Stijlemans B, De Baetselier P, Caljon G, Van Den Abbeele J, Van Ginderachter JA and Magez S: Nanobodies as tools to understand, diagnose, and treat African trypanosomiasis. Front Immunol. 8:7242017. View Article : Google Scholar : PubMed/NCBI
39	Hassanzadeh-Ghassabeh G, Devoogdt N, De Pauw P, Vincke C and Muyldermans S: Nanobodies and their potential applications. Nanomedicine (Lond). 8:1013–1026. 2013. View Article : Google Scholar
40	Arezumand R, Alibakhshi A, Ranjbari J, Ramazani A and Muyldermans S: Nanobodies as novel agents for targeting angio-genesis in solid cancers. Front Immunol. 8:17462017. View Article : Google Scholar
41	Van Heeke G, Allosery K, De Brabandere V, De Smedt T, Detalle L and de Fougerolles A: Nanobodies^® as inhaled biotherapeutics for lung diseases. Pharmacol Ther. 169:47–56. 2017. View Article : Google Scholar
42	Massa S, Xavier C, Muyldermans S and Devoogdt N: Emerging site-specific bioconjugation strategies for radioimmunotracer development. Expert Opin Drug Deliv. 13:1149–1163. 2016. View Article : Google Scholar : PubMed/NCBI
43	Massa S, Vikani N, Betti C, Ballet S, Vanderhaegen S, Steyaert J, Descamps B, Vanhove C, Bunschoten A, van Leeuwen FW, et al: Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: A versatile strategy for multiple molecular imaging modalities. Contrast Media Mol Imaging. 11:328–339. 2016. View Article : Google Scholar : PubMed/NCBI
44	Oliveira S, Heukers R, Sornkom J, Kok RJ, van Bergen EN and Henegouwen PM: Targeting tumors with nanobodies for cancer imaging and therapy. J Control Release. 172:607–617. 2013. View Article : Google Scholar : PubMed/NCBI
45	Iezzi ME, Policastro L, Werbajh S, Podhajcer O and Canziani GA: Single-domain antibodies and the promise of modular targeting in cancer imaging and treatment. Front Immunol. 9:2732018. View Article : Google Scholar : PubMed/NCBI
46	Hu Y, Liu C and Muyldermans S: Nanobody-based delivery systems for diagnosis and targeted tumor therapy. Front Immunol. 8:14422017. View Article : Google Scholar : PubMed/NCBI
47	Beghein E and Gettemans J: Nanobody technology: A versatile toolkit for microscopic imaging, protein-protein interaction analysis, and protein function exploration. Front Immunol. 8:7712017. View Article : Google Scholar : PubMed/NCBI
48	Menzel S, Rissiek B, Haag F, Goldbaum FA and Koch-Nolte F: The art of blocking ADP-ribosyltransferases (ARTs): Nanobodies as experimental and therapeutic tools to block mammalian and toxin ARTs. FEBS J. 280:3543–3550. 2013. View Article : Google Scholar : PubMed/NCBI
49	Unger M, Eichhoff AM, Schumacher L, Strysio M, Menzel S, Schwan C, Alzogaray V, Zylberman V, Seman M, Brandner J, et al: Selection of nanobodies that block the enzy-matic and cytotoxic activities of the binary clostridium difficile toxin CDT. Sci Rep. 5:78502015. View Article : Google Scholar
50	Mars A, Bouhaouala-Zahar B and Raouafi N: Ultrasensitive sensing of androctonus australis hector scorpion venom toxins in biological fluids using an electrochemical graphene quantum dots/nanobody-based platform. Talanta. 190:182–187. 2018. View Article : Google Scholar : PubMed/NCBI
51	Singh A, Pasha SK, Manickam P and Bhansali S: Single-domain antibody based thermally stable electrochemical immunosensor. Biosens Bioelectron. 83:162–168. 2016. View Article : Google Scholar : PubMed/NCBI
52	Zhu Z, Shi L, Feng H and Zhou HS: Single domain antibody coated gold nanoparticles as enhancer for Clostridium difficile toxin detection by electrochemical impedance immunosensors. Bioelectrochemistry. 101:153–158. 2015. View Article : Google Scholar
53	Li G, Zhu M, Ma L, Yan J, Lu X, Shen Y and Wan Y: Generation of small single domain nanobody binders for sensitive detection of testosterone by electrochemical impedance spectroscopy. ACS Appl Mater Interfaces. 8:13830–13839. 2016. View Article : Google Scholar : PubMed/NCBI
54	Li H, Sun Y, Elseviers J, Muyldermans S, Liu S and Wan Y: A nanobody-based electrochemiluminescent immunosensor for sensitive detection of human procalcitonin. Analyst. 139:3718–3721. 2014. View Article : Google Scholar : PubMed/NCBI
55	Liu X, Wen Y, Wang W, Zhao Z, Han Y, Tang K and Wang D: Nanobody-based electrochemical competitive immunosensor for the detection of AFB₁ through AFB₁-HCR as signal amplifier. Mikrochim Acta. 187:3522020. View Article : Google Scholar
56	Liu A, Yin K, Mi L, Ma M, Liu Y, Li Y, Wei W, Zhang Y and Liu S: A novel photoelectrochemical immunosensor by integration of nanobody and ZnO nanorods for sensitive detection of nucleoside diphosphatase kinase-A. Anal Chim Acta. 973:82–90. 2017. View Article : Google Scholar : PubMed/NCBI
57	Zhou Q, Li G, Zhang Y, Zhu M, Wan Y and Shen Y: Highly selective and sensitive electrochemical immunoassay of Cry1C using nanobody and π-π stacked graphene oxide/thionine assembly. Anal Chem. 88:9830–9836. 2016. View Article : Google Scholar : PubMed/NCBI
58	Steeland S, Puimège L, Vandenbroucke RE, Van Hauwermeiren F, Haustraete J, Devoogdt N, Hulpiau P, Leroux-Roels G, Laukens D, Meuleman P, et al: Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1. J Biol Chem. 290:4022–4037. 2015. View Article : Google Scholar :
59	Kazemi-Lomedasht F, Pooshang-Bagheri K, Habibi-Anbouhi M, Hajizadeh-Safar E, Shahbazzadeh D, Mirzahosseini H and Behdani M: In vivo immunotherapy of lung cancer using cross-species reactive vascular endothelial growth factor nano-bodies. Iran J Basic Med Sci. 20:489–496. 2017.PubMed/NCBI
60	Salvador JP, Vilaplana L and Marco MP: Nanobody: Outstanding features for diagnostic and therapeutic applications. Anal Bioanal Chem. 411:1703–1713. 2019. View Article : Google Scholar : PubMed/NCBI
61	De Munter S, Van Parys A, Bral L, Ingels J, Goetgeluk G, Bonte S, Pille M, Billiet L, Weening K, Verhee A, et al: Rapid and effective generation of nanobody based CARs using PCR and gibson assembly. Int J Mol Sci. 21:8832020. View Article : Google Scholar :
62	Ren W, Li Z, Xu Y, Wan D, Barnych B, Li Y, Tu Z, He Q, Fu J and Hammock BD: One-step ultrasensitive bioluminescent enzyme immunoassay based on nanobody/nanoluciferase fusion for detection of aflatoxin B₁ in cereal. J Agric Food Chem. 67:5221–5229. 2019. View Article : Google Scholar : PubMed/NCBI
63	Allegra A, Innao V, Gerace D, Vaddinelli D, Allegra AG and Musolino C: Nanobodies and cancer: Current status and new perspectives. Cancer Invest. 36:221–237. 2018. View Article : Google Scholar : PubMed/NCBI
64	De Genst E, Chan PH, Pardon E, Hsu SD, Kumita JR, Christodoulou J, Menzer L, Chirgadze DY, Robinson CV, Muyldermans S, et al: A nanobody binding to non-amyloido-genic regions of the protein human lysozyme enhances partial unfolding but inhibits amyloid fibril formation. J Phys Chem B. 117:13245–13258. 2013. View Article : Google Scholar : PubMed/NCBI
65	Gonzalez-Sapienza G, Rossotti MA and Tabares-da Rosa S: Single-domain antibodies as versatile affinity reagents for analytical and diagnostic applications. Front Immunol. 8:9772017. View Article : Google Scholar : PubMed/NCBI
66	Akazawa-Ogawa Y, Uegaki K and Hagihara Y: The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies. J Biochem. 159:111–121. 2016. View Article : Google Scholar :
67	Goldman ER, Liu JL, Zabetakis D and Anderson GP: Enhancing stability of camelid and shark single domain antibodies: An overview. Front Immunol. 8:8652017. View Article : Google Scholar : PubMed/NCBI
68	Kunz P, Zinner K, Mücke N, Bartoschik T, Muyldermans S and Hoheisel JD: The structural basis of nanobody unfolding reversibility and thermoresistance. Sci Rep. 8:79342018. View Article : Google Scholar : PubMed/NCBI
69	Schumacher D, Helma J, Schneider AFL, Leonhardt H and Hackenberger CPR: Nanobodies: Chemical functionalization strategies and intracellular applications. Angew Chem Int Ed Engl. 57:2314–2333. 2018. View Article : Google Scholar :
70	Wang Y, Fan Z, Shao L, Kong X, Hou X, Tian D, Sun Y, Xiao Y and Yu L: Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications. Int J Nanomedicine. 11:3287–3303. 2016. View Article : Google Scholar : PubMed/NCBI
71	Jovčevska I and Muyldermans S: The therapeutic potential of nanobodies. BioDrugs. 34:11–26. 2020. View Article : Google Scholar
72	Zottel A, Jovčevska I, Šamec N, Mlakar J, Šribar J, Križaj I, Skoblar Vidmar M and Komel R: Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration. Ther Adv Med Oncol. 12:17588359209153022020. View Article : Google Scholar : PubMed/NCBI
73	Peyron I, Kizlik-Masson C, Dubois MD, Atsou S, Ferrière S, Denis CV, Lenting PJ, Casari C and Christophe OD: Camelid-derived single-chain antibodies in hemostasis: Mechanistic, diagnostic, and therapeutic applications. Res Pract Thromb Haemost. 4:1087–1110. 2020. View Article : Google Scholar : PubMed/NCBI
74	Kijanka M, Dorresteijn B, Oliveira S and van Bergen en Henegouwen PM: Nanobody-based cancer therapy of solid tumors. Nanomedicine (Lond). 10:161–174. 2015. View Article : Google Scholar
75	Huen J, Yan Z, Iwashkiw J, Dubey S, Gimenez MC, Ortiz ME, Patel SV, Jones MD, Riazi A, Terebiznik M, et al: A novel single domain antibody targeting FliC flagellin of salmonella enterica for effective inhibition of host cell invasion. Front Microbiol. 10:26652019. View Article : Google Scholar :
76	Zavrtanik U, Lukan J, Loris R, Lah J and Hadži S: Structural basis of epitope recognition by heavy-chain camelid antibodies. J Mol Biol. 430:4369–4368. 2018. View Article : Google Scholar : PubMed/NCBI
77	Lauwereys M, Arbabi Ghahroudi M, Desmyter A, Kinne J, Hölzer W, De Genst E, Wyns L and Muyldermans S: Potent enzyme inhibitors derived from dromedary heavy-chain anti-bodies. EMBO J. 17:3512–3520. 1998. View Article : Google Scholar : PubMed/NCBI
78	Arbabi-Ghahroudi M: Camelid single-domain antibodies: Historical perspective and future outlook. Front Immunol. 8:15892017. View Article : Google Scholar : PubMed/NCBI
79	Muruganandam A, Tanha J, Narang S and Stanimirovic D: Selection of phage-displayed llama single-domain antibodies that transmigrate across human blood-brain barrier endothelium. FASEB J. 16:240–242. 2002. View Article : Google Scholar : PubMed/NCBI
80	Abulrob A, Sprong H, Van Bergen en Henegouwen P and Stanimirovic D: The blood-brain barrier transmigrating single domain antibody: Mechanisms of transport and antigenic epitopes in human brain endothelial cells. J Neurochem. 95:1201–1214. 2005. View Article : Google Scholar : PubMed/NCBI
81	Menzel S, Schwarz N, Haag F and Koch-Nolte F: Nanobody-based biologics for modulating purinergic signaling in inflammation and immunity. Front Pharmacol. 9:2662018. View Article : Google Scholar : PubMed/NCBI
82	Zhu M, Hu Y, Li G, Ou W, Mao P, Xin S and Wan Y: Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2. Nanoscale Res Lett. 9:5282014. View Article : Google Scholar : PubMed/NCBI
83	Zarschler K, Witecy S, Kapplusch F, Foerster C and Stephan H: High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli. Microb Cell Fact. 12:972013. View Article : Google Scholar : PubMed/NCBI
84	He T, Zhu J, Nie Y, Hu R, Wang T, Li P, Zhang Q and Yang Y: Nanobody technology for mycotoxin detection in the field of food safety: Current status and prospects. Toxins (Basel). 10:1802018. View Article : Google Scholar
85	Detalle L, Stohr T, Palomo C, Piedra PA, Gilbert BE, Mas V, Millar A, Power UF, Stortelers C, Allosery K, et al: Generation and characterization of ALX-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection. Antimicrob Agents Chemother. 60:6–13. 2015. View Article : Google Scholar : PubMed/NCBI
86	Sheng Y, Wang K, Lu Q, Ji P, Liu B, Zhu J, Liu Q, Sun Y, Zhang J, Zhou EM and Zhao Q: Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against newcastle disease virus in the immunoassay. J Nanobiotechnology. 17:352019. View Article : Google Scholar : PubMed/NCBI
87	Wang L, Liu X, Zhu X, Wang L, Wang W, Liu C, Cui H, Sun M and Gao B: Generation of single-domain antibody multimers with three different self-associating peptides. Protein Eng Des Sel. 26:417–423. 2013. View Article : Google Scholar : PubMed/NCBI
88	Behdani M, Zeinali S, Karimipour M, Khanahmad H, Schoonooghe S, Aslemarz A, Seyed N, Moazami-Godarzi R, Baniahmad F, Habibi-Anbouhi M, et al: Development of VEGFR2-specific nanobody pseudomonas exotoxin A conjugated to provide efficient inhibition of tumor cell growth. N Biotechnol. 30:205–209. 2013. View Article : Google Scholar
89	Sadeghnezhad G, Romão E, Bernedo-Navarro R, Massa S, Khajeh K, Muyldermans S and Hassania S: Identification of new DR5 agonistic nanobodies and generation of multivalent nanobody constructs for cancer treatment. Int J Mol Sci. 20:48182019. View Article : Google Scholar :
90	Huet HA, Growney JD, Johnson JA, Li J, Bilic S, Ostrom L, Zafari M, Kowal C, Yang G, Royo A, et al: Multivalent nano-bodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction. Mabs. 6:1560–1570. 2014. View Article : Google Scholar
91	Liu W, Song H, Chen Q, Yu J, Xian M, Nian R and Feng D: Recent advances in the selection and identification of antigen-specific nanobodies. Mol Immunol. 96:37–47. 2018. View Article : Google Scholar : PubMed/NCBI
92	Wagner HJ, Wehrle S, Weiss E, Cavallari M and Weber W: A two-step approach for the design and generation of nanobodies. Int J Mol Sci. 19:34442018. View Article : Google Scholar :
93	Yan J, Wang P, Zhu M, Li G, Romão E, Xiong S and Wan Y: Characterization and applications of nanobodies against human procalcitonin selected from a novel naïve Nanobody phage display library. J Nanobiotechnology. 13:332015. View Article : Google Scholar
94	Itoh K, Reis AH, Hayhurst A and Sokol SY: Isolation of nanobodies against xenopus embryonic antigens using immune and nonimmune phage display libraries. PLoS One. 14:e02160832019. View Article : Google Scholar
95	Yan J, Li G, Hu Y, Ou W and Wan Y: Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications. J Transl Med. 12:3432014. View Article : Google Scholar : PubMed/NCBI
96	Cui Y, Li D, Morisseau C, Dong JX, Yang J, Wan D, Rossotti MA, Gee SJ, González-Sapienza GG and Hammock BD: Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase. Anal Bioanal Chem. 407:7275–7283. 2015. View Article : Google Scholar : PubMed/NCBI
97	Gong X, Zhu M, Li G, Lu X and Wan Y: Specific determination of influenza H7N2 virus based on biotinylated single-domain antibody from a phage-displayed library. Anal Biochem. 500:66–72. 2016. View Article : Google Scholar
98	Vincke C, Gutiérrez C, Wernery U, Devoogdt N, Hassanzadeh-Ghassabeh G and Muyldermans S: Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods Mol Biol. 907:145–176. 2012. View Article : Google Scholar : PubMed/NCBI
99	Behar G, Sibéril S, Groulet A, Chames P, Pugnière M, Boix C, Sautès-Fridman C, Teillaud JL and Baty D: Isolation and characterization of anti-FcgammaRIII (CD16) llama single-domain antibodies that activate natural killer cells. Protein Eng Des Sel. 21:1–10. 2018. View Article : Google Scholar
100	Maussang D, Mujić-Delić A, Descamps FJ, Stortelers C, Vanlandschoot P, Stigter-van Walsum M, Vischer HF, van Roy M, Vosjan M, Gonzalez-Pajuelo M, et al: Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo. J Biol Chem. 288:29562–29572. 2013. View Article : Google Scholar : PubMed/NCBI
101	Farajpour Z, Rahbarizadeh F, Kazemi B and Ahmadvand D: A nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment. Biochem Biophys Res Commun. 446:132–136. 2014. View Article : Google Scholar : PubMed/NCBI
102	Kim HJ, McCoy MR, Majkova Z, Dechant JE, Gee SJ, Tabares-da Rosa S, González-Sapienza GG and Hammock BD: Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay. Anal Chem. 84:1165–1171. 2012. View Article : Google Scholar :
103	Li K, Zettlitz KA, Lipianskaya J, Zhou Y, Marks JD, Mallick P, Reiter RE and Wu AM: A fully human scFv phage display library for rapid antibody fragment reformatting. Protein Eng Des Sel. 28:307–316. 2015. View Article : Google Scholar : PubMed/NCBI
104	Doshi R, Chen BR, Vibat CR, Huang N, Lee CW and Chang G: In vitro nanobody discovery for integral membrane protein targets. Sci Rep. 4:67602014. View Article : Google Scholar : PubMed/NCBI
105	Ferrari D, Garrapa V, Locatelli M and Bolchi A: A novel nanobody scaffold optimized for bacterial expression and suitable for the construction of ribosome display libraries. Mol Biotechnol. 62:43–55. 2020. View Article : Google Scholar
106	Yau KY, Groves MA, Li S, Sheedy C, Lee H, Tanha J, MacKenzie CR, Jermutus L and Hall JC: Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library. J Immunol Methods. 281:161–175. 2003. View Article : Google Scholar : PubMed/NCBI
107	Bencurova E, Pulzova L, Flachbartova Z and Bhide M: A rapid and simple pipeline for synthesis of mRNA-ribosome-V(H)H complexes used in single-domain antibody ribosome display. Mol Biosyst. 11:1515–1524. 2015. View Article : Google Scholar : PubMed/NCBI
108	McMahon C, Baier AS, Pascolutti R, Wegrecki M, Zheng S, Ong JX, Erlandson SC, Hilger D, Rasmussen SGF, Ring AM, et al: Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat Struct Mol Biol. 25:289–296. 2018. View Article : Google Scholar : PubMed/NCBI
109	Uchański T, Zögg T, Yin J, Yuan D, Wohlkönig A, Fischer B, Rosenbaum DM, Kobilka BK, Pardon E and Steyaert J: An improved yeast surface display platform for the screening of nanobody immune libraries. Sci Rep. 9:3822019. View Article : Google Scholar
110	Salema V and Fernández LÁ: Escherichia coli surface display for the selection of nanobodies. Microb Biotechnol. 10:1468–1484. 2017. View Article : Google Scholar : PubMed/NCBI
111	Salema V, Marín E, Martínez-Arteaga R, Ruano-Gallego D, Fraile S, Margolles Y, Teira X, Gutierrez C, Bodelón G and Fernández LÁ: Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two β-domains of opposite topologies. PLoS One. 8:e751262013. View Article : Google Scholar
112	Salema V, Mañas C, Cerdán L, Piñero-Lambea C, Marín E, Roovers RC, Van Bergen En Henegouwen PM and Fernández LÁ: High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display. MAbs. 8:1286–1301. 2016. View Article : Google Scholar : PubMed/NCBI
113	Tang J, Li J, Zhu X, Yu Y, Chen D, Yuan L, Gu Z, Zhang X, Qi L, Gong Z, et al: Novel CD7-specific nanobody-based immunotoxins potently enhanced apoptosis of CD7-positive malignant cells. Oncotarget. 7:34070–34083. 2016. View Article : Google Scholar : PubMed/NCBI
114	Hassel JC, Heinzerling L, Aberle J, Bähr O, Eigentler TK, Grimm MO, Grünwald V, Leipe J, Reinmuth N, Tietze JK, et al: Combined immune checkpoint blockade (anti-PD-1/anti-CTLA-4): Evaluation and management of adverse drug reactions. Cancer Treat Rev. 57:36–49. 2017. View Article : Google Scholar : PubMed/NCBI
115	Gupta A, De Felice KM, Loftus EV Jr and Khanna S: Systematic review: Colitis associated with anti-CTLA-4 therapy. Aliment Pharmacol Ther. 42:406–417. 2015. View Article : Google Scholar : PubMed/NCBI
116	Savoia P, Astrua C and Fava P: Ipilimumab (Anti-Ctla-4 Mab) in the treatment of metastatic melanoma: Effectiveness and toxicity management. Hum Vaccin Immunother. 12:1092–1101. 2016. View Article : Google Scholar : PubMed/NCBI
117	Gao X and McDermott DF: Ipilimumab in combination with nivolumab for the treatment of renal cell carcinoma. Expert Opin Biol Ther. 18:947–957. 2018. View Article : Google Scholar : PubMed/NCBI
118	Tang Z, Mo F, Liu A, Duan S, Yang X, Liang L, Hou X, Yin S, Jiang X, Vasylieva N, et al: A nanobody against cytotoxic T-lymphocyte associated antigen-4 increases the anti-tumor effects of specific CD8⁺ T cells. J Biomed Nanotechnol. 15:2229–2239. 2019. View Article : Google Scholar : PubMed/NCBI
119	Mahoney KM, Freeman GJ and McDermott DF: The next immune-checkpoint inhibitors: PD-1/PD-L1 blockade in melanoma. Clin Ther. 37:764–782. 2015. View Article : Google Scholar : PubMed/NCBI
120	Broos K, Lecocq Q, Keersmaecker B, Raes G, Corthals J, Lion E, Thielemans K, Devoogdt N, Keyaerts M and Breckpot K: Single domain antibody-mediated blockade of programmed death-ligand 1 on dendritic cells enhances CD8 T-cell activation and cytokine production. Vaccines (Basel). 7:852019. View Article : Google Scholar
121	Fang T, Li R, Li Z, Cho J, Guzman JS, Kamm RD and Ploegh HL: Remodeling of the tumor microenvironment by a chemokine/Anti-PD-L1 nanobody fusion protein. Mol Pharm. 16:2838–2844. 2019. View Article : Google Scholar : PubMed/NCBI
122	Zhang F, Wei H, Wang X, Bai Y, Wang P, Wu J, Jiang X, Wang Y, Cai H, Xu T and Zhou A: Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade. Cell Discov. 3:170042017. View Article : Google Scholar : PubMed/NCBI
123	Xian Z, Ma L, Zhu M, Li G, Gai J, Chang Q, Huang Y, Ju D and Wan Y: Blocking the PD-1-PD-L1 axis by a novel PD-1 specific nanobody expressed in yeast as a potential therapeutic for immunotherapy. Biochem Biophys Res Commun. 519:267–273. 2019. View Article : Google Scholar : PubMed/NCBI
124	Li S, Jiang K, Wang T, Zhang W, Shi M, Chen B and Hua Z: Nanobody against PDL1. Biotechnol Lett. 42:727–736. 2020. View Article : Google Scholar : PubMed/NCBI
125	Liu J, Zhang S, Hu Y, Yang Z, Li J, Liu X, Deng L, Wang Y, Zhang X, Jiang T and Lu X: Targeting PD-1 and Tim-3 path-ways to reverse CD8 T-cell exhaustion and enhance ex vivo T-cell responses to autologous dendritic/tumor vaccines. J Immunother. 39:171–180. 2016. View Article : Google Scholar : PubMed/NCBI
126	Liu JF, Ma SR, Mao L, Bu LL, Yu GT, Li YC, Huang CF, Deng WW, Kulkarni AB, Zhang WF and Sun ZJ: T-cell immu-noglobulin mucin 3 blockade drives an antitumor immune response in head and neck cancer. Mol Oncol. 11:235–247. 2017. View Article : Google Scholar : PubMed/NCBI
127	Chang X, Lu X, Guo J and Teng GJ: Interventional therapy combined with immune checkpoint inhibitors: Emerging opportunities for cancer treatment in the era of immunotherapy. Cancer Treat Rev. 74:49–60. 2019. View Article : Google Scholar : PubMed/NCBI
128	Ascione A, Arenaccio C, Mallano A, Flego M, Gellini M, Andreotti M, Fenwick C, Pantaleo G, Vella S and Federico M: Development of a novel human phage display-derived anti-LAG3 scFv antibody targeting CD8⁺ T lymphocyte exhaustion. BMC Biotechnol. 19:672019. View Article : Google Scholar
129	Homayouni V, Ganjalikhani-Hakemi M, Rezaei A, Khanahmad H, Behdani M and Lomedasht FK: Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3). Iran J Basic Med Sci. 19:1201–1208. 2016.PubMed/NCBI
130	Ma LL, Zhu M, Li GH, Li YF, Gai JW and Wan YK: Construction and screening of phage display library for TIM-3 nanobody. Acta Pharmaceutica Sinica. 53:388–395. 2018.
131	Long L, Zhang X, Chen F, Pan Q, Phiphatwatchara P, Zeng Y and Chen H: The promising immune checkpoint LAG-3: From tumor microenvironment to cancer immunotherapy. Genes Cancer. 9:176–189. 2018. View Article : Google Scholar
132	Everett KL, Kraman M, Wollerton FPG, Zimarino C, Kmiecik K, Gaspar M, Pechouckova S, Allen NL, Doody JF and Tuna M: Generation of Fcabs targeting human and murine LAG-3 as building blocks for novel bispecific antibody therapeutics. Methods. 154:60–69. 2019. View Article : Google Scholar
133	Puhr HC and Ilhan-Mutlu A: New emerging targets in cancer immunotherapy: The role of LAG3. ESMO Open. 4:e0004822019. View Article : Google Scholar : PubMed/NCBI
134	Dmitriev OY, Lutsenko S and Muyldermans S: Nanobodies as probes for protein dynamics in vitro and in cells. J Biol Chem. 291:3767–3775. 2016. View Article : Google Scholar :
135	Chanier T and Chames P: Nanobody engineering: Toward next generation immunotherapies and immunoimaging of cancer. Antibodies (Basel). 8:132019. View Article : Google Scholar
136	Lecocq Q, Zeven K, De Vlaeminck Y, Martens S, Massa S, Goyvaerts C, Raes G, Keyaerts M, Breckpot K and Devoogdt N: Noninvasive imaging of the immune checkpoint LAG-3 using nanobodies, from development to pre-clinical use. Biomolecules. 9:5482019. View Article : Google Scholar :
137	Lv G, Sun X, Qiu L, Sun Y, Li K, Liu Q, Zhao Q, Qin S and Lin J: PET imaging of tumor PD-L1 expression with a highly specific nonblocking single-domain antibody. J Nucl Med. 61:117–122. 2020. View Article : Google Scholar :
138	Broos K, Keyaerts M, Lecocq Q, Renmans D, Nguyen T, Escors D, Liston A, Raes G, Breckpot K and Devoogdt N: Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers. Oncotarget. 8:41932–41946. 2017. View Article : Google Scholar : PubMed/NCBI
139	Broos K, Lecocq Q, Xavier C, Bridoux J, Nguyen TT, Corthals J, Schoonooghe S, Lion E, Raes G, Keyaerts M, et al: Evaluating a single domain antibody targeting human PD-L1 as a nuclear imaging and therapeutic agent. Cancers (Basel). 11:8722019. View Article : Google Scholar
140	Wan R, Liu A, Hou X, Lai Z, Li J, Yang N, Tan J, Mo F, Hu Z, Yang X, et al: Screening and antitumor effect of an anti-CTLA-4 nanobody. Oncol Rep. 39:511–518. 2018.
141	Wang W, Hou X, Yang X, Liu A, Tang Z, Mo F, Yin S and Lu X: Highly sensitive detection of CTLA-4-positive T-cell subgroups based on nanobody and fluorescent carbon quantum dots. Oncol Lett. 18:109–116. 2019.PubMed/NCBI