Biology subjects

Pishesha, N.

Publications and source records attributed to Pishesha, N..

4 recordsLinked to original sources

bioRxiv2024

Nanobody-targeted conditional antimicrobial therapeutics

Conditional therapeutics that rely on disease microenvironment-specific triggers for activation are a promising strategy to improve therapeutic cargos. Among the investigated triggers, protease activity is used most often, because of its dysregulation in several diseases. How to optimally fine-tune protease activation for different therapeutic cargos remains a challenge. Here, we designed nanobody-targeted conditional antimicrobial therapeutics to deliver a model therapeutic peptide and protein to the site of bacterial infection. We explored several parameters that influence proteolytic activation. We report the use of targeting nanobodies to enhance the activation of therapeutics that are otherwise activated inefficiently, despite extensive optimization of the cleavable linker. Specifically, pairing of Ly6G/C or ADAM10-targeting nanobodies with ADAM10-cleavable linkers improved activation via proximity-enabled reactivity. More broadly, this optimization framework provides a guideline for the development of conditional therapeutics to treat various diseases where protease activity is dysregulated.

bioengineering↗

bioRxiv2022

Humanized antibody potently neutralizes all SARS-CoV-2 variants by a novel mechanism

SARS-CoV-2 Omicron variants have generated a world-wide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of antibodies induced by vaccination. Here, we describe the SARS-CoV-2 neutralizing SP1-77 antibody that was generated from a humanized mouse model with a single human VH1-2 and V{kappa}1-33-associated with immensely diverse complementarity-determining-region-3 (CDR3) sequences. SP1-77 potently and broadly neutralizes SARS-CoV-2 variants of concern and binds the SARS-CoV-2 spike protein receptor-binding-domain (RBD) via a novel-CDR3-based mode. SP1-77 does not block RBD-binding to the ACE2-receptor or endocytosis step of viral entry, but rather blocks membrane fusion. Our findings provide the first mechanistic insight into how a non-ACE2 blocking antibody potently neutralizes SARS-CoV-2, which may inform strategies for designing vaccines that robustly neutralize current and future SARS-CoV-2 variants.

immunology↗

bioRxiv2020

Engineered Red Blood Cells Carrying PCSK9 Inhibitors Persistently Lower LDL and Prevent Obesity

Low plasma levels of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) are associated with decreased low-density lipoprotein (LDL) cholesterol and a reduced risk of cardiovascular disease. PCSK9 binds to the epidermal growth factor-like repeat A (EGFA) domain of LDL receptors (LDLR), very low-density lipoprotein receptors (VLDLR), apolipoprotein E receptor 2 (ApoER2), and lipoprotein receptor-related protein 1 (LRP1) and accelerates their degradation, thus acting as a key regulator of lipid metabolism. Antibody and RNAi - based PCSK9 inhibitor treatments lower cholesterol and prevent cardiovascular incidents in patients, but their high cost hampers market penetration. We sought to develop a safe, long-term and one-time solution to treat hyperlipidemia. We created a cDNA encoding a chimeric protein in which the extracellular N-terminus of glycophorin A was fused to the LDLR EGFA domain and introduced this gene into mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). Following transplantation into irradiated mice, the animals produced red blood cells (RBCs) with the EGFA domain (EGFA-GPA RBCs) displayed on their surface. These animals showed significantly reduced plasma PCSK9 (66.5% decrease) and reduced LDL levels (40% decrease) for as long as 12 months post-transplantation. Furthermore, the EGFA-GPA mice remained lean for life and maintained normal body weight under high-fat diet. Hematopoietic stem cell gene therapy can generate red blood cells expressing an EGFA - glycophorin A chimeric protein as a practical and long-term strategy for treating chronic hyperlipidemia and obesity.

bioengineering↗

bioRxiv2020

Erythrocyte-targeted immunomodulatory antigens enabled by in vivo selection of D-peptides

Targeting of antigens to erythrocytes can be used to selectively mitigate their immunogenicity, but the methods to equip a variety of cargoes with erythrocyte-targeting properties are limited. Here we identified a D-peptide that targets murine erythrocytes and decreases anti-drug antibody responses when conjugated to the protective antigen from Bacillus anthracis, a protein of therapeutic interest. The D-peptide likewise decreases inflammatory anti-ovalbumin (OVA) CD8+ T cell responses when attached to a peptide antigen derived from OVA. To discover this targeting ligand, we leveraged mass spectrometry to decode a randomized D-peptide library selected in mice, extending the application of synthetic libraries to in vivo affinity selections.

bioengineering↗