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@article{pmid40411427,

title = {Synthetic antibodies targeting EphA2 induce diverse signaling-competent clusters with differential activation},

author = {Jarrett J Adams and Heather A Bruce and Suryasree Subramania and Lynda Ploder and Julia Garcia and Isabelle Pot and Levi L Blazer and Alexander U Singer and Sachdev S Sidhu},

doi = {10.1002/pro.70145},

issn = {1469-896X},

year  = {2025},

date = {2025-06-01},

urldate = {2025-06-01},

journal = {Protein Sci},

volume = {34},

number = {6},

pages = {e70145},

abstract = {The receptor tyrosine kinase EphA2 interacts with ephrin (Efn) ligands to mediate bi-directional signals that drive cellular sorting processes during tissue development. In the context of various cancers, EphA2 can also drive invasive metastatic disease and represents an important target for cancer therapeutics. Natural Efn ligands sterically seed intertwined EphA2 clusters capable of recruiting intracellular kinases to mediate trans-phosphorylation. Synthetic proteins, such as antibodies (Abs), can mimic Efn ligands to trigger EphA2 signaling, leading to receptor internalization and degradation, and enabling intracellular delivery of conjugated drugs. Furthermore, Abs are capable of recruiting EphA2 into clusters distinct from those seeded by Efn. We developed three synthetic Abs targeting distinct EphA2 domains and determined the paratope valency necessary for agonist or antagonist properties of each of the three epitopes. Structural modeling of monovalent Fabs in complex with EphA2 elucidated competitive and non-competitive mechanisms of inhibition of EphA2 canonical signaling. Likewise, modeling of clusters induced by bivalent IgGs elucidated multiple signaling-competent EphA2 clusters capable of triggering a continuum of signaling strengths and provided insights into the requirement for multimerization of EphA2 to trigger phosphorylation. Our study shows how different agonist clusters lead to distinct kinase recruitment efficiencies to modify phosphotyrosine signal strength, and provides a panel of anti-EphA2 Abs as reagents for the development of therapeutics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40384600,

title = {Molecular characterization of a synthetic neutralizing antibody targeting p67 of Theileria parva},

author = {Shane Miersch and Alex U Singer and Chao Chen and Fred Fellouse and Anupriya Gopalsamy and Luciano Silva E Costa and Anna Lacasta and Hannah Chege and Naomi Chege and Vishvanath Nene and Sachdev S Sidhu},

doi = {10.1002/pro.70153},

issn = {1469-896X},

year  = {2025},

date = {2025-06-01},

urldate = {2025-06-01},

journal = {Protein Sci},

volume = {34},

number = {6},

pages = {e70153},

abstract = {The Theileria parva sporozoite surface antigen p67 is a target of the bovine humoral immune response that generates antibodies capable of providing protection against subsequent infection. As a result, p67 has been the subject of efforts aimed at the development of an anti-sporozoite subunit vaccine. Previous studies have identified neutralizing epitopes in the N- and C-terminal regions of the full-length protein and shown that immunization with a C-terminal fragment of p67 (p67C) alone is capable of eliciting protection. To identify additional neutralizing epitopes in p67C, selections were conducted against it using a phage-displayed synthetic antibody library. An antibody that neutralized the sporozoite in vitro was identified, and the crystal structure of a Fab:peptide complex was elucidated. Mutagenesis studies aimed at validating and further characterizing the Fab:peptide interaction identified critical residues involved in binding and neutralization. This study also validates distinct epitopes for previously reported neutralizing antibodies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40329529,

title = {Locking CBL TKBD in its native conformation presents a novel therapeutic opportunity in mutant CBL-dependent leukemia},

author = {Syed Feroj Ahmed and Jayanthi Anand and Wei Zhang and Lori Buetow and Loveena Rishi and Louise Mitchell and Jonathan Bohlen and Sergio Lilla and Gary J Sibbet and Colin Nixon and Amrita Patel and Karolina A Majorek and Sara Zanivan and Jacinta C Bustamante and Sachdev S Sidhu and Karen Blyth and Danny T Huang},

doi = {10.1016/j.ymthe.2025.04.042},

issn = {1525-0024},

year  = {2025},

date = {2025-05-01},

urldate = {2025-05-01},

journal = {Mol Ther},

abstract = {Casitas B-lineage lymphoma (CBL) is an E3 ubiquitin ligase critical for negatively regulating receptor protein tyrosine kinases (RTKs). Deleterious CBL mutants lose E3 activity, but act as adaptors that gain function to cause myeloproliferative neoplasms. Currently, there is no targeted treatment available for patients with CBL mutant-dependent disorders. By combining phage-display technology and structure-based optimization, we discovered CBLock, a nanomolar affinity peptide inhibitor, that binds the substrate-binding site of CBL's tyrosine kinase binding domain (TKBD). CBLock disrupts the interaction between CBL mutants and RTKs, thereby impairing RTK-mediated priming of adaptor function of CBL mutants and downstream signaling. Notably, CBLock binds TKBD without inducing conformational changes, thereby preserving its ligand-free native conformation. In contrast, when CBL binds RTK substrates, TKBD undergoes a conformational change. Maintaining the native CBL TKBD conformation was crucial for CBLock to inhibit proliferation, induce cell-cycle arrest, and promote apoptosis in leukemia cells harboring CBL mutations. In a mouse xenograft model of acute myeloid leukemia (AML), CBLock reduced tumor burden and improved survival rate. Moreover, CBLock inhibited the proliferation of cells derived from patients with CBL mutations. Therefore, inhibiting CBL TKBD in its native state presents a promising therapeutic opportunity in targeting mutant CBL-dependent leukemia.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}