{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,7]],"date-time":"2025-11-07T05:19:43Z","timestamp":1762492783645,"version":"build-2065373602"},"reference-count":35,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T00:00:00Z","timestamp":1762300800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"the National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82373777","82473846"],"award-info":[{"award-number":["82373777","82473846"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Immune checkpoint inhibitors (ICIs) targeting PD-L1 and CD47 are clinically limited by severe off-target toxicities. To address this issue, immunotherapeutic prodrug strategies have been developed, aimed at preventing antibodies from binding to targets in healthy tissues and thereby reducing systemic toxicity. Existing strategies include prodrug technologies that mask the active sites of antibodies via peptide or polyethylene glycol (PEG) modification\u2014yet these approaches also cause antibodies to lose their targeting ability. Herein, we propose an antibody prodrug strategy (termed FA-PEG-S-Ab) with active targeting capability. By modifying antibodies with folate-PEG-disulfide and PEG-disulfide linkages, we developed two novel prodrugs: FA-PEG-S-Atz (PD-L1-blocking prodrug) and FA-PEG-S-Hu5 (CD47-blocking prodrug). This strategy functions through two key steps: first, folate binding to folate receptor \u03b1 (FR\u03b1)-mediated tumor-specific targeting enables the prodrugs to accumulate specifically in tumor tissues; subsequently, the high concentration of glutathione (GSH) in the tumor microenvironment (TME) specifically cleaves the disulfide bonds, removing the PEG shield, releasing the antibody, and restoring the antibody\u2019s antigen-binding activity. In vitro experiments confirmed that the modified antibody prodrug FA-PEG-S-Hu5 exhibits high affinity for FR\u03b1 (KD = 4.02 \u00d7 10\u22129 M) and effectively masks the antibody\u2019s binding activity (KD from 1.05 \u00d7 10\u221211 M to 2.10 \u00d7 10\u22128 M). Following activation by GSH in the TME, this masking effect is reversed, and the antibody regains its binding affinity (KD = 2.14 \u00d7 10\u221210 M). Crucially, FA-PEG-S-Hu5 completely eliminates hemolytic toxicity. This \u201cfolate targeting delivery + TME activation\u201d prodrug strategy is expected to provide a new solution for addressing the off-target toxicities of conventional ICIs.<\/jats:p>","DOI":"10.3390\/molecules30214292","type":"journal-article","created":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T07:58:05Z","timestamp":1762329485000},"page":"4292","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Glutathione-Responsive Folate-Targeted Prodrugs: Tumor-Specific PD-L1 and CD47 Blockade"],"prefix":"10.3390","volume":"30","author":[{"given":"Jianfeng","family":"Wang","sequence":"first","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lianqi","family":"Liu","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dian","family":"Xiao","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fei","family":"Xie","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinbo","family":"Zhou","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100850, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1038\/s41572-020-0160-6","article-title":"Immune-related adverse events of checkpoint inhibitors","volume":"6","author":"Brahmer","year":"2020","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1714","DOI":"10.1200\/JCO.2017.77.6385","article-title":"Management of Immune-Related Adverse Events in Patients Treated With Immune Checkpoint Inhibitor Therapy: American Society of Clinical Oncology Clinical Practice Guideline","volume":"36","author":"Brahmer","year":"2018","journal-title":"J. Clin. Oncol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2455","DOI":"10.1056\/NEJMoa1200694","article-title":"Safety and activity of anti-PD-L1 antibody in patients with advanced cancer","volume":"366","author":"Brahmer","year":"2012","journal-title":"N. Engl. J. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2051","DOI":"10.1126\/science.288.5473.2051","article-title":"Role of CD47 as a marker of self on red blood cells","volume":"288","author":"Oldenborg","year":"2000","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Jajosky, R.P., Covington, M.L., Liu, J., Chai, L., Zerra, P.E., Chonat, S., Stowell, S.R., and Arthur, C.M. (2025). CD47 regulates antigen modulation and red blood cell clearance following an incompatible transfusion. Front. Immunol., 16.","DOI":"10.3389\/fimmu.2025.1548548"},{"key":"ref_6","unstructured":"Gilead Sciences (2017). A Phase 1b Trial of Magrolimab Monotherapy or Magrolimab in Combination with Azacitidine in Patients with Hematological Malignancies."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"7009","DOI":"10.1200\/JCO.2019.37.15_suppl.7009","article-title":"The first-in-class anti-CD47 antibody Hu5F9-G4 is active and well tolerated alone or with azacitidine in AML and MDS patients: Initial phase 1b results","volume":"37","author":"Sallman","year":"2019","journal-title":"J. Clin. Oncol."},{"key":"ref_8","unstructured":"ALX Oncology Inc (2020). A Phase 1\/2 Study of Evorpacept (ALX148) in Combination with Azacitidine in Patients with Higher Risk Myelodysplastic Syndrome (MDS) (ASPEN-02)."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"49","DOI":"10.32604\/or.2023.042383","article-title":"Opportunities and challenges of CD47-targeted therapy in cancer immunotherapy","volume":"32","author":"Chen","year":"2023","journal-title":"Oncol. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.addr.2016.03.008","article-title":"Tumor penetrating peptides for improved drug delivery","volume":"110\u2013111","author":"Ruoslahti","year":"2017","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2007","DOI":"10.1172\/JCI92284","article-title":"Tumor-penetrating peptide enhances transcytosis of silicasome-based chemotherapy for pancreatic cancer","volume":"127","author":"Liu","year":"2017","journal-title":"J. Clin. Investig."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1038\/nrd1033","article-title":"Effect of pegylation on pharmaceuticals","volume":"2","author":"Harris","year":"2003","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1038\/s41467-020-14471-1","article-title":"Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy","volume":"11","author":"Sharma","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1067","DOI":"10.1016\/j.addr.2004.01.001","article-title":"Distribution, functionality and gene regulation of folate receptor isoforms: Implications in targeted therapy","volume":"56","author":"Elnakat","year":"2004","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"100917","DOI":"10.1016\/j.currproblcancer.2022.100917","article-title":"Folate Receptor as a Biomarker and Therapeutic Target in Solid Tumors","volume":"47","author":"Young","year":"2023","journal-title":"Curr. Probl. Cancer"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"52553","DOI":"10.18632\/oncotarget.9651","article-title":"Targeting folate receptor alpha for cancer treatment","volume":"7","author":"Cheung","year":"2016","journal-title":"Oncotarget"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"414","DOI":"10.18632\/oncotarget.519","article-title":"Folate receptor alpha expression in lung cancer: Diagnostic and prognostic significance","volume":"3","author":"Yu","year":"2012","journal-title":"Oncotarget"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1038\/s41523-020-0147-1","article-title":"Folate receptor alpha expression associates with improved disease-free survival in triple negative breast cancer patients","volume":"6","author":"Norton","year":"2020","journal-title":"npj Breast Cancer"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1186\/2193-1801-1-22","article-title":"Folate receptor alpha (FRA) expression in breast cancer: Identification of a new molecular subtype and association with triple negative disease","volume":"1","author":"Somers","year":"2012","journal-title":"Springerplus"},{"key":"ref_20","unstructured":"Immunogen, Inc (2019). MIRASOL: A Randomized, Open-label, Phase 3 Study of Mirvetuximab Soravtansine vs. Investigator\u2019s Choice of Chemotherapy in Platinum-Resistant, Advanced High-Grade Epithelial Ovarian, Primary Peritoneal, or Fallopian Tube Cancers With High Folate Receptor-Alpha Expression."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1731","DOI":"10.1038\/s41589-025-01924-1","article-title":"Polyvalent folate receptor-targeting chimeras for degradation of membrane proteins","volume":"21","author":"Xiao","year":"2025","journal-title":"Nat. Chem. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"8695","DOI":"10.1038\/s41467-024-52685-9","article-title":"Development of folate receptor targeting chimeras for cancer selective degradation of extracellular proteins","volume":"15","author":"Zhou","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1038\/s41571-020-0339-5","article-title":"Exploiting the folate receptor \u03b1 in oncology","volume":"17","author":"Scaranti","year":"2020","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1038\/nm0297-177","article-title":"Interstitial pH and pO2 gradients in solid tumors in vivo: High-resolution measurements reveal a lack of correlation","volume":"3","author":"Helmlinger","year":"1997","journal-title":"Nat. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1016\/j.ijpharm.2018.07.061","article-title":"Glutathione-responsive self-delivery nanoparticles assembled by curcumin dimer for enhanced intracellular drug delivery","volume":"549","author":"Zhang","year":"2018","journal-title":"Int. J. Pharm."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1021\/acsnano.8b06400","article-title":"Glutathione-Responsive Prodrug Nanoparticles for Effective Drug Delivery and Cancer Therapy","volume":"13","author":"Ling","year":"2019","journal-title":"ACS Nano"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Zhang, R., Liu, T., Li, W., Ma, Z., Pei, P., Zhang, W., Yang, K., and Tao, Y. (2022). Tumor microenvironment-responsive BSA nanocarriers for combined chemo\/chemodynamic cancer therapy. J. Nanobiotechnol., 20.","DOI":"10.1186\/s12951-022-01442-5"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"128880","DOI":"10.1016\/j.cej.2021.128880","article-title":"Glutathione-mediated nanomedicines for cancer diagnosis and therapy","volume":"426","author":"Ding","year":"2021","journal-title":"Chem. Eng. J."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"10721","DOI":"10.1021\/am402860v","article-title":"Reduction-cleavable polymeric vesicles with efficient glutathione-mediated drug release behavior for reversing drug resistance","volume":"5","author":"Ren","year":"2013","journal-title":"ACS Appl. Mater. Interfaces"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2407","DOI":"10.1021\/bm2005164","article-title":"Redox-responsive polyphosphate nanosized assemblies: A smart drug delivery platform for cancer therapy","volume":"12","author":"Liu","year":"2011","journal-title":"Biomacromolecules"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2954","DOI":"10.1039\/C6SC00170J","article-title":"Construction of homogeneous antibody-drug conjugates using site-selective protein chemistry","volume":"7","author":"Akkapeddi","year":"2016","journal-title":"Chem. Sci."},{"key":"ref_32","unstructured":"Graham Collins (2016). A Phase 1b\/2 Trial of Hu5F9-G4 in Combination with Rituximab or Rituximab + Chemotherapy in Patients with Relapsed\/Refractory B-cell Non-Hodgkin\u2019s Lymphoma."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e005517","DOI":"10.1136\/jitc-2022-005517","article-title":"Ligufalimab, a novel anti-CD47 antibody with no hemagglutination demonstrates both monotherapy and combo antitumor activity","volume":"10","author":"Qu","year":"2022","journal-title":"J. Immunother. Cancer"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1200\/JCO.18.02018","article-title":"First-in-Human, First-in-Class Phase I Trial of the Anti-CD47 Antibody Hu5F9-G4 in Patients With Advanced Cancers","volume":"37","author":"Sikic","year":"2019","journal-title":"J. Clin. Oncol."},{"key":"ref_35","first-page":"A288","article-title":"266 AK117, a CD47 blocking antibody with robust macrophage activation without red blood cell hemagglutination","volume":"9","author":"Huang","year":"2021","journal-title":"J. Immunother. Cancer"}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/21\/4292\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,7]],"date-time":"2025-11-07T05:16:25Z","timestamp":1762492585000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/21\/4292"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,5]]},"references-count":35,"journal-issue":{"issue":"21","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["molecules30214292"],"URL":"https:\/\/doi.org\/10.3390\/molecules30214292","relation":{},"ISSN":["1420-3049"],"issn-type":[{"type":"electronic","value":"1420-3049"}],"subject":[],"published":{"date-parts":[[2025,11,5]]}}}