{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T15:38:48Z","timestamp":1776440328597,"version":"3.51.2"},"reference-count":28,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2021,6,8]],"date-time":"2021-06-08T00:00:00Z","timestamp":1623110400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003130","name":"Fonds Wetenschappelijk Onderzoek","doi-asserted-by":"publisher","award":["G085620N"],"award-info":[{"award-number":["G085620N"]}],"id":[{"id":"10.13039\/501100003130","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Immune checkpoint inhibitors targeting the programmed cell death-1 (PD-1) and its ligand PD-L1 have proven to be efficient cancer therapies in a subset of patients. From all the patients with various cancer types, only 20% have a positive response. Being able to distinguish patients that do express PD-1\/PD-L1 from patients that do not allows patients to benefit from a more personalized and efficient treatment of tumor lesion(s). Expression of PD-1 and PD-L1 is typically assessed via immunohistochemical detection in a tumor biopsy. However, this method does not take in account the expression heterogeneity within the lesion, nor the possible metastasis. To visualize whole-body PD-L1 expression by PET imaging, we developed a nanobody-based radio-immunotracer targeting PD-L1 site-specifically labeled with gallium-68. The cysteine-tagged nanobody was site-specifically conjugated with a maleimide (mal)-NOTA chelator and radiolabeling was tested at different nanobody concentrations and temperatures. Affinity and specificity of the tracer, referred to as [68Ga]Ga-NOTA-mal-hPD-L1 Nb, were assayed by surface plasmon resonance and on PD-L1POS or PD-L1NEG 624-MEL cells. Xenografted athymic nude mice bearing 624-MEL PD-L1POS or PD-L1NEG tumors were injected with the tracer and ex vivo biodistribution was performed 1 h 20 min post-injection. Ideal 68Ga-labeling conditions were found at 50 \u00b0C for 15 min. [68Ga]Ga-NOTA-mal-hPD-L1 Nb was obtained in 80 \u00b1 5% DC-RCY with a RCP &gt; 99%, and was stable in injection buffer and human serum up to 3 h (&gt;99% RCP). The in vitro characterization showed that the NOTA-functionalized Nb retained its affinity and specificity. Ex vivo biodistribution revealed a tracer uptake of 1.86 \u00b1 0.67% IA\/g in the positive tumors compared with 0.42 \u00b1 0.04% IA\/g in the negative tumors. Low background uptake was measured in the other organs and tissues, except for the kidneys and bladder, due to the expected excretion route of Nbs. The data obtained show that the site-specific 68Ga-labeled NOTA-mal-hPD-L1 Nb is a promising PET radio-immunotracer due to its ease of production, stability and specificity for PD-L1.<\/jats:p>","DOI":"10.3390\/ph14060550","type":"journal-article","created":{"date-parts":[[2021,6,8]],"date-time":"2021-06-08T21:16:58Z","timestamp":1623187018000},"page":"550","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":30,"title":["Site-Specific Radiolabeling of a Human PD-L1 Nanobody via Maleimide\u2013Cysteine Chemistry"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8288-5302","authenticated-orcid":false,"given":"Dora Mugoli","family":"Chigoho","sequence":"first","affiliation":[{"name":"In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Medical Imaging Department (MIMA), Vrije Universiteit Brussel, 1090 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2627-0880","authenticated-orcid":false,"given":"Quentin","family":"Lecocq","sequence":"additional","affiliation":[{"name":"Laboratory for Molecular and Cellular Therapy (LMCT), Department of Biomedical Sciences, Vrije Universiteit Brussel, 1090 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robin Maximilian","family":"Awad","sequence":"additional","affiliation":[{"name":"Laboratory for Molecular and Cellular Therapy (LMCT), Department of Biomedical Sciences, Vrije Universiteit Brussel, 1090 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4331-3480","authenticated-orcid":false,"given":"Karine","family":"Breckpot","sequence":"additional","affiliation":[{"name":"Laboratory for Molecular and Cellular Therapy (LMCT), Department of Biomedical Sciences, Vrije Universiteit Brussel, 1090 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9220-4833","authenticated-orcid":false,"given":"Nick","family":"Devoogdt","sequence":"additional","affiliation":[{"name":"In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Medical Imaging Department (MIMA), Vrije Universiteit Brussel, 1090 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9997-4571","authenticated-orcid":false,"given":"Marleen","family":"Keyaerts","sequence":"additional","affiliation":[{"name":"In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Medical Imaging Department (MIMA), Vrije Universiteit Brussel, 1090 Brussels, Belgium"},{"name":"Department of Nuclear Medicine, UZ Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vicky","family":"Caveliers","sequence":"additional","affiliation":[{"name":"In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Medical Imaging Department (MIMA), Vrije Universiteit Brussel, 1090 Brussels, Belgium"},{"name":"Department of Nuclear Medicine, UZ Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9006-1074","authenticated-orcid":false,"given":"Catarina","family":"Xavier","sequence":"additional","affiliation":[{"name":"In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Medical Imaging Department (MIMA), Vrije Universiteit Brussel, 1090 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8895-9763","authenticated-orcid":false,"given":"Jessica","family":"Bridoux","sequence":"additional","affiliation":[{"name":"In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Medical Imaging Department (MIMA), Vrije Universiteit Brussel, 1090 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,6,8]]},"reference":[{"key":"ref_1","first-page":"115","article-title":"Innovations en oncologie m\u00e9dicale\u2014que retenir de","volume":"137","author":"Baurain","year":"2018","journal-title":"Louvain Med."},{"key":"ref_2","first-page":"5","article-title":"d Aspects fondamentaux","volume":"95","author":"Vuagnat","year":"2018","journal-title":"JNDES"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/j.canlet.2014.12.047","article-title":"The role of IL-21 in immunity and cancer","volume":"358","author":"Davis","year":"2015","journal-title":"Cancer Lett."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1477","DOI":"10.1038\/nm955","article-title":"Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice","volume":"9","author":"Okazaki","year":"2003","journal-title":"Nat. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1016\/j.csbj.2019.03.006","article-title":"Application of PD-1 Blockade in Cancer Immunotherapy","volume":"17","author":"Wu","year":"2019","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"ref_6","unstructured":"FDA (2021). Hematology\/Oncology (Cancer) Approvals & Safety Notifications, FDA. Available online: https:\/\/www.fda.gov\/drugs\/resources-information-approved-drugs\/hematologyoncology-cancer-approvals-safety-notifications."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2749","DOI":"10.3389\/fimmu.2018.02749","article-title":"Promises and Pitfalls in the Use of PD-1\/PD-L1 Inhibitors in Multiple Myeloma","volume":"9","author":"Oliva","year":"2018","journal-title":"Front. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1097\/PPO.0000000000000303","article-title":"Mechanisms of Resistance to PD-1 and PD-L1 Blockade","volume":"24","author":"Nowicki","year":"2018","journal-title":"Cancer J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1186\/s12943-018-0864-3","article-title":"Biomarkers for predicting efficacy of PD-1\/PD-L1 inhibitors","volume":"17","author":"Yi","year":"2018","journal-title":"Mol. Cancer"},{"key":"ref_10","first-page":"819324","article-title":"Advance of Molecular Imaging Technology and Targeted Imaging Agent in Imaging and Therapy","volume":"2014","author":"Chen","year":"2014","journal-title":"BioMed Res. Int."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1007\/s00259-012-2298-2","article-title":"PET imaging with a [68Ga]gallium-labelled PSMA ligand for the diagnosis of prostate cancer: Biodistribution in humans and first evaluation of tumour lesions","volume":"40","author":"Malcher","year":"2013","journal-title":"Eur. J. Nucl. Med. Mol. Imaging"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Broos, K., Lecocq, Q., Xavier, C., Bridoux, J., Nguyen, T.T., Corthals, J., Schoonooghe, S., Lion, E., Raes, G., and Keyaerts, M. (2019). Evaluating a Single Domain Antibody Targeting Human PD-L1 as a Nuclear Imaging and Therapeutic Agent. Cancers, 11.","DOI":"10.3390\/cancers11060872"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Bridoux, J., Broos, K., Lecocq, Q., Debie, P., Martin, C., Ballet, S., Raes, G., Neyt, S., Vanhove, C., and Breckpot, K. (2020). Anti-Human PD-L1 Nanobody for Immuno-PET Imaging: Validation of a Conjugation Strategy for Clinical Translation. Biomolecules, 10.","DOI":"10.3390\/biom10101388"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1021\/bc500111t","article-title":"Site-Specific Labeling of Cysteine-Tagged Camelid Single-Domain Antibody-Fragments for Use in Molecular Imaging","volume":"25","author":"Massa","year":"2014","journal-title":"Bioconjug. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1149","DOI":"10.1080\/17425247.2016.1178235","article-title":"Emerging site-specific bioconjugation strategies for radioimmunotracer development","volume":"13","author":"Massa","year":"2016","journal-title":"Expert Opin. Drug Deliv."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1002\/chem.201803174","article-title":"Bioconjugation with Maleimides: A Useful Tool for Chemical Biology","volume":"25","author":"Ravasco","year":"2019","journal-title":"Chem. A Eur. J."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3273","DOI":"10.1074\/jbc.M806889200","article-title":"General Strategy to Humanize a Camelid Single-Domain Antibody and Identification of a Universal Humanized Nanobody Scaffold","volume":"284","author":"Vincke","year":"2009","journal-title":"J. Biol. Chem."},{"key":"ref_18","unstructured":"Van Ginderachter, J., Raes, G., Devoogdt, N., Keyaerts, M., Lecocq, Q., Xavier, C., Breckpot, K., Broos, K., and Bridoux, J. (2019). Human pd-l1-Binding Immunoglobulins. (WO 2019166622A1), Available online: https:\/\/patentscope.wipo.int\/search\/en\/detail.jsf?docId=WO2019166622."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"24828","DOI":"10.1039\/C7RA04606E","article-title":"Use of a next generation maleimide in combination with THIOMAB\u2122 antibody technology delivers a highly stable, potent and near homogeneous THIOMAB\u2122 antibody-drug conjugate (TDC)","volume":"7","author":"Nunes","year":"2017","journal-title":"RSC Adv."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1038\/nature16057","article-title":"Novel antibody\u2013antibiotic conjugate eliminates intracellular S. aureus","volume":"527","author":"Lehar","year":"2015","journal-title":"Nature"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"12462","DOI":"10.1073\/pnas.0705347104","article-title":"[64Cu-NOTA-8-Aoc-BBN(7-14)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues","volume":"30","author":"Prasanphanich","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"894","DOI":"10.1021\/bc100470x","article-title":"Evaluation of a Maleimido Derivative of NOTA for Site-Specific Labeling of Affibody Molecules","volume":"22","author":"Tolmachev","year":"2011","journal-title":"Bioconjug. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1970","DOI":"10.1074\/jbc.M111.242818","article-title":"Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments","volume":"287","author":"GGovaert","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"776","DOI":"10.2967\/jnumed.112.111021","article-title":"Synthesis, Preclinical Validation, Dosimetry, and Toxicity of 68Ga-NOTA-Anti-HER2 Nanobodies for iPET Imaging of HER2 Receptor Expression in Cancer","volume":"54","author":"Xavier","year":"2013","journal-title":"J. Nucl. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1021\/bc5005262","article-title":"Long-Term Stabilization of Maleimide\u2013Thiol Conjugates","volume":"26","author":"Fontaine","year":"2015","journal-title":"Bioconjug. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1038\/nbt.2108","article-title":"Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates","volume":"30","author":"Shen","year":"2012","journal-title":"Nat. Biotechnol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2433","DOI":"10.1096\/fj.10-180331","article-title":"Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer","volume":"25","author":"Vaneycken","year":"2011","journal-title":"FASEB J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2807","DOI":"10.1128\/AAC.45.10.2807-2812.2001","article-title":"\u03b2-Lactamase Inhibitors Derived from Single-Domain Antibody Fragments Elicited in the Camelidae","volume":"45","author":"Conrath","year":"2001","journal-title":"Antimicrob. Agents Chemother."}],"container-title":["Pharmaceuticals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8247\/14\/6\/550\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:12:12Z","timestamp":1760163132000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8247\/14\/6\/550"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,6,8]]},"references-count":28,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2021,6]]}},"alternative-id":["ph14060550"],"URL":"https:\/\/doi.org\/10.3390\/ph14060550","relation":{},"ISSN":["1424-8247"],"issn-type":[{"value":"1424-8247","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,8]]}}}