{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:52:33Z","timestamp":1782280353142,"version":"3.54.5"},"posted":{"date-parts":[[2025,8,2]]},"group-title":"Pharmacology and Toxicology","reference-count":41,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,8,2]],"date-time":"2025-08-02T00:00:00Z","timestamp":1754092800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100000062","name":"National Institute of Diabetes and Digestive and Kidney Diseases","doi-asserted-by":"crossref","award":["R01DK137299"],"award-info":[{"award-number":["R01DK137299"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,8,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  CD28 is a key T cell co-stimulatory receptor implicated in antitumor immunity and immune-related disorders, yet no small molecule modulators of CD28 have reached clinical development. Here, we report the discovery and characterization of small molecule CD28 antagonists identified through affinity selection-mass spectrometry (AS-MS). Subsequent catalog-based structure\u2013activity relationship (SAR) optimization led to the identification of two lead compounds,\n                  <jats:bold>5MS-5<\/jats:bold>\n                  and\n                  <jats:bold>19MS-5<\/jats:bold>\n                  , which exhibit direct CD28 binding and potent inhibition of CD28\u2013B7 interactions in cellular reporter assays. In vitro pharmacokinetic profiling demonstrated favorable solubility, metabolic stability, and permeability, alongside low off-target liabilities. Functionally, both compounds suppressed cytokine production in primary human T cells co-cultured with tumor spheroids and human epithelial tissues, validating their ability to inhibit CD28-driven immune activation in physiologically relevant models. These findings establish\n                  <jats:bold>5MS-5<\/jats:bold>\n                  and\n                  <jats:bold>19MS-5<\/jats:bold>\n                  as promising CD28 inhibitors and provide a foundation for developing orally bioavailable immunomodulators targeting T cell co-stimulation.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Table of Contents artwork<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"667814v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.1101\/2025.07.31.667814","type":"posted-content","created":{"date-parts":[[2025,8,2]],"date-time":"2025-08-02T16:35:12Z","timestamp":1754152512000},"source":"Crossref","is-referenced-by-count":7,"title":["Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation"],"prefix":"10.64898","author":[{"given":"Saurabh","family":"Upadhyay","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sungwoo","family":"Cho","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hossam","family":"Nada","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Moustafa T.","family":"Gabr","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025080515500448000_2025.07.31.667814v1.1","doi-asserted-by":"crossref","first-page":"102283","DOI":"10.1016\/j.coi.2023.102283","article-title":"Immune checkpoint receptors in autoimmunity","volume":"80","year":"2023","journal-title":"Curr Opin Immunol"},{"key":"2025080515500448000_2025.07.31.667814v1.2","doi-asserted-by":"publisher","DOI":"10.1097\/00007890-199705270-00016"},{"key":"2025080515500448000_2025.07.31.667814v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccell.2024.11.008"},{"issue":"10","key":"2025080515500448000_2025.07.31.667814v1.4","doi-asserted-by":"crossref","first-page":"110947","DOI":"10.1016\/j.isci.2024.110947","article-title":"Artificial antigen-presenting cell system reveals CD28\u2019s role in modulating T cell functions during human immunodeficiency virus infection","volume":"27","year":"2024","journal-title":"iScience"},{"key":"2025080515500448000_2025.07.31.667814v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.bmcl.2025.130310"},{"key":"2025080515500448000_2025.07.31.667814v1.6","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.87.13.5031"},{"issue":"8","key":"2025080515500448000_2025.07.31.667814v1.7","doi-asserted-by":"crossref","first-page":"2461","DOI":"10.4049\/jimmunol.147.8.2461","article-title":"CD28 delivers a costimulatory signal involved in antigen-specific IL-2 production by human T cells","volume":"147","year":"1991","journal-title":"J Immunol"},{"key":"2025080515500448000_2025.07.31.667814v1.8","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1103231"},{"key":"2025080515500448000_2025.07.31.667814v1.9","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.176.6.3306"},{"key":"2025080515500448000_2025.07.31.667814v1.10","doi-asserted-by":"crossref","unstructured":"Kaur, K. ; Chen, P. C. ; Ko, M. W. ; Mei, A. ; Chovatiya, N. ; Huerta-Yepez, S. ; Ni, W. ; Mackay, S. ; Zhou, J. ; Maharaj, D. ; et al. The Potential Role of Cytotoxic Immune Effectors in Induction, Progression and Pathogenesis of Amyotrophic Lateral Sclerosis (ALS). Cells 2022, 11 (21).","DOI":"10.3390\/cells11213431"},{"key":"2025080515500448000_2025.07.31.667814v1.11","first-page":"1081163","article-title":"Selective CD28 blockade impacts T cell differentiation during homeostatic reconstitution following lymphodepletion","volume":"13","year":"2022","journal-title":"Front Immunol"},{"issue":"2","key":"2025080515500448000_2025.07.31.667814v1.12","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1007\/s12253-011-9471-y","article-title":"Dysregulated expression of both the costimulatory CD28 and inhibitory CTLA-4 molecules in PB T cells of advanced cervical cancer patients suggests systemic immunosuppression related to disease progression","volume":"18","year":"2012","journal-title":"Pathol Oncol Res"},{"key":"2025080515500448000_2025.07.31.667814v1.13","doi-asserted-by":"crossref","unstructured":"Sobhani, N. ; Tardiel-Cyril, D. R. ; Chai, D. ; Generali, D. ; Li, J. R. ; Vazquez-Perez, J. ; Lim, J. M. ; Morris, R. ; Bullock, Z. N. ; Davtyan, A. ; et al. Artificial intelligence-powered discovery of small molecules inhibiting CTLA-4 in cancer. BJC Rep 2024, 2.","DOI":"10.1038\/s44276-023-00035-5"},{"issue":"5","key":"2025080515500448000_2025.07.31.667814v1.14","doi-asserted-by":"crossref","first-page":"4083","DOI":"10.1021\/acs.jmedchem.3c02362","article-title":"Discovery of Highly Potent Small-Molecule PD-1\/PD-L1 Inhibitors with a Novel Scaffold for Cancer Immunotherapy","volume":"67","year":"2024","journal-title":"J Med Chem"},{"key":"2025080515500448000_2025.07.31.667814v1.15","doi-asserted-by":"crossref","unstructured":"Calvo-Barreiro, L. ; Gabr, M. T. TRIC-Based High-Throughput Screening Enables the Discovery of Small Molecule CD28 Binders. bioRxiv 2025, 2025.05.27.656499.","DOI":"10.1101\/2025.05.27.656499"},{"key":"2025080515500448000_2025.07.31.667814v1.16","doi-asserted-by":"crossref","unstructured":"Calvo-Barreiro, L. ; Nada, H. ; Upadhyay, S. ; Gabr, M. T. Surface Plasmon Resonance (SPR)-Based Workflow for High-Throughput Discovery of CD28-Targeted Small Molecules. bioRxiv 2025, 2025.2006.2012.659248.","DOI":"10.1101\/2025.06.12.659248"},{"key":"2025080515500448000_2025.07.31.667814v1.17","doi-asserted-by":"publisher","DOI":"10.4103\/0975-1483.66810"},{"key":"2025080515500448000_2025.07.31.667814v1.18","doi-asserted-by":"publisher","DOI":"10.1111\/ajt.12964"},{"key":"2025080515500448000_2025.07.31.667814v1.19","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1300470"},{"key":"2025080515500448000_2025.07.31.667814v1.20","doi-asserted-by":"publisher","DOI":"10.1124\/jpet.115.227249"},{"key":"2025080515500448000_2025.07.31.667814v1.21","doi-asserted-by":"crossref","unstructured":"Porciello, N. ; Kunkl, M. ; Tuosto, L. CD28 between tolerance and autoimmunity: the side effects of animal models. F1000Res 2018, 7.","DOI":"10.12688\/f1000research.14046.1"},{"issue":"670","key":"2025080515500448000_2025.07.31.667814v1.22","doi-asserted-by":"crossref","first-page":"eabn1082","DOI":"10.1126\/scitranslmed.abn1082","article-title":"CD22-targeted CD28 bispecific antibody enhances antitumor efficacy of odronextamab in refractory diffuse large B cell lymphoma models","volume":"14","year":"2022","journal-title":"Sci Transl Med"},{"key":"2025080515500448000_2025.07.31.667814v1.23","doi-asserted-by":"publisher","DOI":"10.1158\/2326-6066.CIR-20-0253"},{"key":"2025080515500448000_2025.07.31.667814v1.24","doi-asserted-by":"publisher","DOI":"10.1186\/S12943-024-02212-7"},{"issue":"6","key":"2025080515500448000_2025.07.31.667814v1.25","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1016\/j.immuni.2019.11.003","article-title":"PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways","volume":"51","year":"2019","journal-title":"Immunity"},{"key":"2025080515500448000_2025.07.31.667814v1.26","doi-asserted-by":"publisher","DOI":"10.1002\/cmdc.202400545"},{"issue":"11","key":"2025080515500448000_2025.07.31.667814v1.27","doi-asserted-by":"crossref","first-page":"103760","DOI":"10.1016\/j.drudis.2023.103760","article-title":"Affinity selection mass spectrometry speeding drug discovery","volume":"28","year":"2023","journal-title":"Drug Discov Today"},{"key":"2025080515500448000_2025.07.31.667814v1.28","doi-asserted-by":"publisher","DOI":"10.15252\/emmm.202114850"},{"key":"2025080515500448000_2025.07.31.667814v1.29","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.5c00009"},{"key":"2025080515500448000_2025.07.31.667814v1.30","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2023.128403"},{"key":"2025080515500448000_2025.07.31.667814v1.31","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.0c00899"},{"key":"2025080515500448000_2025.07.31.667814v1.32","doi-asserted-by":"publisher","DOI":"10.1021\/acsmedchemlett.3c00054"},{"key":"2025080515500448000_2025.07.31.667814v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/j.slasd.2023.04.003"},{"issue":"11","key":"2025080515500448000_2025.07.31.667814v1.34","doi-asserted-by":"crossref","first-page":"1884","DOI":"10.1021\/acsmedchemlett.4c00350","article-title":"From Virtual Screens to Cellular Target Engagement: New Small Molecule Ligands for the Immune Checkpoint LAG-3","volume":"15","year":"2024","journal-title":"ACS Med Chem Lett"},{"issue":"42","key":"2025080515500448000_2025.07.31.667814v1.35","doi-asserted-by":"crossref","first-page":"eadq5540","DOI":"10.1126\/sciadv.adq5540","article-title":"Small molecules from antibody pharmacophores (SMAbPs) as a hit identification workflow for immune checkpoints","volume":"10","year":"2024","journal-title":"Sci. Adv"},{"key":"2025080515500448000_2025.07.31.667814v1.36","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.3c00960"},{"key":"2025080515500448000_2025.07.31.667814v1.37","doi-asserted-by":"publisher","DOI":"10.1002\/cmdc.202300305"},{"issue":"9","key":"2025080515500448000_2025.07.31.667814v1.38","doi-asserted-by":"crossref","first-page":"1767","DOI":"10.1039\/D3MD00150D","article-title":"First-in-class small molecule inhibitors of ICOS\/ICOSL interaction as a novel class of immunomodulators","volume":"14","year":"2023","journal-title":"RSC Med Chem"},{"key":"2025080515500448000_2025.07.31.667814v1.39","doi-asserted-by":"publisher","DOI":"10.1002\/cmdc.202400545"},{"key":"2025080515500448000_2025.07.31.667814v1.40","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.0c07276"},{"key":"2025080515500448000_2025.07.31.667814v1.41","unstructured":"Corporation, P. CD28 Blockade Bioassay, JA6101. Promega Corporation 2025. (acccessed 2025)."}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.07.31.667814","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:00:32Z","timestamp":1782277232000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.07.31.667814"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,2]]},"references-count":41,"URL":"https:\/\/doi.org\/10.1101\/2025.07.31.667814","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1021\/acs.jmedchem.5c02136","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2025,8,2]]},"subtype":"preprint"}}