Biology subjects

Zoppi, G.

Publications and source records attributed to Zoppi, G..

3 recordsLinked to original sources

bioRxiv2026

Dissociation kinetics and avidity gate SARS-CoV-2 neutralization by HR2 stem helix antibodies

SARS-CoV-2 evolution has reduced the efficacy of clinical monoclonal antibodies, underscoring the need for therapeutics targeting conserved viral regions. The Spike (S) heptad repeat 2 (HR2) stem helix is highly conserved across SARS-CoV-2 variants and related betacoronaviruses. Although antibodies to this region can neutralize infection, their natural occurrence and evolution remain poorly understood. We previously identified human neutralizing antibodies to a conserved peptide within this region (HR2 coldspot). Here, we show that plasma IgG reactivity to this region remains rare, even after repeated antigen exposure. Longitudinal analysis over 30 months revealed continued somatic hypermutation of HR2-specific antibodies, yet none surpassed the potency or breadth of hr2.016, which emerged shortly after primary infection. Crystal structures of four HR2 stem helix antibodies revealed convergent recognition across distinct antibody lineages. Comparison of hr2.016 with its non-neutralizing clonal relative hr2.086 showed that structural convergence masks distinct binding kinetics. Surface plasmon resonance and molecular dynamics simulations revealed a more stable interaction network for hr2.016, with slower dissociation and prolonged S residence time. Neutralization required the IgG format, supporting an avidity-driven mechanism. Together, these findings define kinetic and avidity constraints governing neutralization at the HR2 stem helix and position hr2.016 as a resilient therapeutic candidate.

immunology↗

bioRxiv2026

Droplet single-cell CRISPR screens identify regulators of T cell-mediated target-cell killing

Cytotoxic CD8 T cells kill target cells through brief cell-cell encounters, but pooled genetic screens cannot readily link perturbations in individual T cells to the fate of the target cells they engage. We developed droplet single-cell CRISPR screening to pair individual primary human CD8 T cells with cancer cells, measure rapid target-cell death, and recover sgRNAs from phenotype-defined droplets. Applied across primary T cells from multiple donors, the platform recovered regulators of T cell receptor signaling, synapse formation, granule exocytosis and cytotoxic differentiation, and identified negative regulators of killing, including established inhibitory nodes such as PTEN, RASA2 and FOXO1, together with AFAP1L2 and components of the mTORC1 pathway. Validation across bispecific engager and TCR-engineered settings showed that selected hits modulate target-cell killing across recognition modalities and tumor models. Unexpectedly, perturbation of RPTOR or RHEB enhanced cytotoxic execution while reducing mTORC1 output, increasing AKT phosphorylation, and attenuating anabolic programs. Transient pharmacologic mTORC1 inhibition reproduced this rapid-killing state and improved antitumor activity after adoptive transfer. These results establish an interaction-resolved pooled genetic strategy for mapping cytotoxicity regulators and reveal that transient modulation of mTORC1 can shift T cells from anabolic growth toward rapid cytotoxic execution to enhance antitumor activity.

immunology↗

bioRxiv2025

AI assisted design of ligands for Lipocalin-2

Lipocalin-2 (LCN2) is an acute-phase glycoprotein whose upregulation is linked to blood- brain-barrier breakdown and neuroinflammation, making it an attractive diagnostic and therapeutic target. We developed an end-to-end, AI-guided workflow to rapidly design de-novo miniproteins that bind LCN2. Backbone scaffolds were generated with RFdiffusion, sequences were optimized with ProteinMPNN, and candidates filtered in silico using a consensus of AlphaFold2 confidence metrics (mean interface pAE < 10) and binding free energy predicted by Prodigy. From an initial library of 10,000 designs, five were expressed and purified from E. coli. Using biolayer interferometry (BLI) we identified MiniP-2 as the lead construct, exhibiting a dissociation constant (Kd) of 4.2 nM. Structural modeling revealed that binding is primarily mediated by backbone hydrogen bonds along with a stabilizing salt bridge between Arg37 of MinP-2 and Asp97 of LCN2. These findings demonstrate that a fully computational generative workflow can yield nanomolar LCN2 binders in a single design-build-test cycle. MinP-2 represents a promising starting point for affinity maturation, structural studies, and in vivo evaluation as an imaging probe or antagonist of LCN2-mediated signaling. Specifically, SPR competition experiments showed that MinP-2 can inhibit LCN2 binding to MMP-9, suggesting its potential to mitigate the pathological effects of this interaction within the central nervous system.

biochemistry↗