Biology subjects

Nicely, N. I.

Publications and source records attributed to Nicely, N. I..

4 recordsLinked to original sources

bioRxiv2026

Computational design of orthogonal TCR α/β interfaces for dual-TCR therapeutics

T-cell receptors (TCRs) recognize peptides presented by MHC, enabling access to intracellular targets that are largely inaccessible to antibodies and difficult to target with small molecules. Despite this potential, their inherent cross-reactivity limits tumor specificity, while single-antigen targeting provides limited coverage of intratumoral heterogeneity. Dual-TCR therapeutics comprising two distinct TCRs could enhance tumor specificity via combinatorial recognition while broadening coverage across heterogeneous antigens. However, practical development of dual-TCR therapeutics has been limited by /{beta} subunit mispairing that prevents efficient production and creates undesired binding properties. Here, we develop orthogonal TCR /{beta} interfaces that prevent subunit mispairing. Using computational multistate design and second-site suppressor strategies implemented in Rosetta, we identified over 250 TCR variants for experimental screening to assess protein stability and pairing fidelity. The top-performing designs achieved approximately 95% correct pairing, as validated by mass spectrometry and X-ray crystallography. Focusing mutations on constant domains and conserved framework regions of variable domains enabled broad applicability across diverse TCRs while preserving antigen recognition. Using these orthogonal interfaces, we developed trispecific T-cell engagers (TriTEs) that target two cancer-testis antigens and CD3 on T cells, demonstrating enhanced potency under dual-antigen engagement (EC50 of 380 fM) while maintaining high activity when targeting cells displaying a single antigen (EC50s of 48 pM and 20 pM). This orthogonal TCR interface technology establishes a generalizable platform for engineering multi-specific immune therapeutics targeting diverse cancer antigens.

bioengineering↗

bioRxiv2025

Structural determination of the HIV-1 Variable Region 3 epitope of antibody 19b

The HIV-1 Envelope (Env) in its pre-receptor "closed" conformation is targeted by broadly neutralizing antibodies (bnAbs), while its receptor-bound "open" conformation, exposes immunodominant epitopes targeted by non-neutralizing antibodies. A human immunoglobin G (IgG) monoclonal antibody (mAb), 19b, binds an Env third variable (V3) loop epitope that is only exposed in the open Env conformation. Despite widespread use of 19b to detect the open Env conformation in immunoassays, its epitope has not yet been structurally defined. Here we determine crystal structures of ligand-free and V3 peptide-bound 19b Fab to visualize details of this interaction. 19b utilizes both its heavy and light chains to interact with the V3 loop. The 5-residue heavy chain complementarity-determining region (CDR H3) facilitates a hydrophobic pocket for V3 residues to associate with. 19b adopts a cradle binding mode with its CDRH1, CDRL2 and CDRL3 mediating interactions with V3 regions that flank the conserved GPGR/Q motif, without making substantial contacts with the GPGR arch region. Our high-resolution structures by elucidating the epitope, binding mode and the structural basis for the broad reactivity of 19b, fill a gap in our knowledge of a reagent that is widely used in immunoassays.

biochemistry↗

bioRxiv2025

Computational Stabilization of the Human VH Germline Repertoire to Enable Conditional Multi-Specific Therapeutic Development

Protein-based biologic therapies, particularly antibody-like therapeutics, have emerged as a major modality to treat nearly all chronic and infectious diseases. Antagonists have dominated the first wave of antibody and antibody-like biologics, whereas agonism has generally been challenging due to systemic activation leading to issues with therapeutic index and problems with pleiotropic activity. Additionally, agonists that use native proteins such as cytokines can be challenging to produce at scale given these proteins evolved to act locally and are not designed for large scale manufacturing. To address these challenges, we generated a biologics platform comprised of stabilized human VH domains (VH-Select) which encompass the entire human germline repertoire with the goal of building multispecific biologics denoted Tentacles that use avidity-based binding to achieve conditional activity directed to specific cell types or tissues. Stabilizing disulfides and point mutations were identified computationally with Rosetta, evaluated in vitro, and combined into designs with 3-5 amino acid substitutions for each of the seven germline families (VH1-VH7). Computational design was also employed to reduce dimerization from both VL and homotypic VH-VH interactions. Optimization of specific sequences improved expression by greater than 600-fold and thermostability by more than 20{degrees}C. Each of the germline variants were screened for low HLA class II binding and incorporated into a library which demonstrated significant improvements in cellular protein production, thereby increasing sequence diversity for screening campaigns. These VH-Select scaffolds are useful for the discovery of novel binders used to build multispecific Tentacles designed for cis-interactions that achieve cell- and tissue-specific activation. As an example of the utility of the platform, we generated a set of Tentacles that use VH-Select binders to conditionally agonize IL2R{gamma}{beta} and 41BB on PD1+, LAG3+, or CD8+ T cells. These Tentacles demonstrate promising manufacturability, antibody-like exposure in vivo, and strong anti-tumor activity in a humanized tumor model. Overall, we believe the incorporation of VH-Select binders into multispecific Tentacles has the potential to create a host of conditionally active biologics to treat various chronic and acute diseases.

bioengineering↗

bioRxiv2025

CCDC32 collaborates with the membrane to assemble the AP-2 clathrin adaptor complex

Cells have evolved a variety of assembly chaperones to aid in the difficult process of forming macromolecular complexes in a crowded cytoplasm. Assembly of adaptor protein complex 2 (AP-2), the primary cargo adaptor in clathrin-mediated endocytosis, is regulated by the chaperones AAGAB and CCDC32, whose deletion causes loss of all AP-2 subunits in vivo. AAGAB and CCDC32 are thought to act sequentially to assemble the AP-2 tetramer from its constituent heterodimers. However, the molecular requirements and structural consequences of CCDC32 interaction with AP-2 are not yet understood. Here, using in vitro reconstitution and integrative structural analysis, we describe the molecular mechanism of CCDC32-mediated AP-2 assembly. First, CCDC32 interacts with the appendage domain of the AP-2 subunit, using the same binding site as canonical endocytic regulators in addition to a novel, yet highly conserved pocket on . CCDC32 contains cargo sorting motifs normally found in trans-membrane cargo and binds to AP-2 heterodimers using canonical cargo-binding sites. Additionally, two amphipathic helices in CCDC32 bind to the /{sigma}2 heterodimer. Surprisingly, in solution, we find that CCDC32 prevents complex assembly and actively disassembles AP-2 tetramers. Inhibition requires the amphipathic helices of CCDC32, which also mediate binding to PIP2-containing membranes. The presence of PIP2-containing membrane stabilizes the final stages of assembly. We propose that the membrane acts as a molecular switch to release inhibitory interactions, allowing for full complex assembly to proceed. Using cryo-EM, we visualize an assembly intermediate that mimics the conformation of AP-2 found in vesicles, with CCDC32 bound at both cargo binding sites and both membrane-binding sites, suggesting that assembly leads to deposition of active complexes on the plasma membrane.

molecular biology↗