Biology subjects

Duty, J. A.

Publications and source records attributed to Duty, J. A..

6 recordsLinked to original sources

bioRxiv2026

A Human Recombinant Antibody Against DRP1 Serine 616 Phosphorylation Enables Detection of BRAFV600E-Associated Mitochondrial Division in Cancer

Mitochondria are dynamic organelles that continuously undergo balanced cycles of fusion and division to meet cellular demands. Mitochondrial division is mediated by Dynamin-Related Protein 1 (DRP1), a cytosolic large GTPase whose phosphorylation at serine 616 (DRP1-S616) promotes its translocation to the outer mitochondrial membrane and organelle division. Dysregulated, mitochondrial division disrupts cellular homeostasis and contributes to disease pathogenesis, including cancer. Our prior work demonstrated that the oncogene-induced mitogen-activated protein kinase (MAPK) pathway constitutively phosphorylates DRP1 at serine 616 (DRP1-S616), which is essential to cellular transformation and correlates with oncogene status in patient tissues. Similarly, DRP1-S616 is subject to pharmacologic control by targeted therapies against oncogenic MAPK signaling. Building upon this foundation, we developed a human recombinant monoclonal antibody with high specificity for DRP1-S616, referred to as 3G11. Using diverse biochemical platforms, we demonstrate the robust utility of 3G11 to detect DRP1-S616 in melanoma cell extracts and isolated organelles. Immunofluorescence revealed that pharmacologic inhibition of oncogenic MAPK signaling reduces DRP1-S616 levels which correlates with mitochondrial hyperfusion; while immunohistochemistry showed that elevated DRP1-S616 expression in human tissues correlates with BRAFV600E disease. Together, these findings establish 3G11 as a specific, versatile, renewable, and cost-effective tool for studying mitochondrial division, with strong potential for clinical applications.

cancer biology↗

bioRxiv2025

Human monoclonal antibodies that target clade 2.3.4.4b H5N1 hemagglutinin

The highly pathogenic avian influenza H5N1 virus clade 2.3.4.4b has been spreading globally since 2022, causing mortality and morbidity in domestic and wild birds and mammals, including infection in humans, raising concerns about its pandemic potential. We aimed to generate a panel of anti-hemagglutinin (HA) human monoclonal antibodies (mAbs) against the H5 protein of clade 2.3.4.4b. H2L2 Harbour Mice(R), which express human immunoglobulin germline genes, were immunized with H5 and N1 recombinant proteins from A/mallard/New York/22-008760-007-original/2022 H5N1 virus, enabling the generation of human chimeric antibodies. Through hybridoma technology, sixteen full human mAbs were generated, most of which showed cross-reactivity against H5 proteins from different virus variants. The functionality of the sixteen mAbs was assessed in vitro using hemagglutination inhibition and microneutralization assays with viruses containing a clade 2.3.4.4b HA. Fourteen out of the sixteen mAbs neutralized the virus in vitro. The mAbs with the strongest hemagglutination inhibition activity also demonstrated greater neutralizing capacity and showed increased protective effects in vivo when administered prophylactically or therapeutically in a murine H5N1 challenge model. Using cryo-electron microscopy, we identified a cross-clonotype conserved motif that bound a hydrophobic groove on the head domain of H5 HA. Akin to mAbs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the coronavirus 2019 (COVID-19) pandemic, these mAbs could serve as important treatments in case of a widespread H5N1 epidemic or pandemic.

microbiology↗

bioRxiv2024

Heterologous immunization strategy developed broadly reactive human monoclonal antibodies against the BK virus

BK polyomavirus (BKV) causes polyomavirus-associated nephropathy (BKV-nephropathy) and polyomavirus-associated hemorrhagic cystitis (BKV-HC) following kidney transplantation and allogeneic hematopoietic stem cell transplantation (HSCT). BKV strains consist of four distinct genotypes (BKV-I, - II, -III, and -IV) with more than 80% of individuals seropositive for the BKV-I genotype, and lower prevalences of infection or co-infection with the other genotypes. BKV-nephropathy occurs widely in immunosuppressed transplant recipients, with the recommended treatment including reduction of immunosuppressive drugs. High serum titers of BKV-neutralizing antibodies and treatment with BKV-neutralizing intravenous immunoglobulin (IVIG) are associated with reduced levels of BKV-DNAemia in kidney transplant recipients, suggesting anti-BKV antibodies can limit viral load. Thus, we set out to generate broadly neutralizing human monoclonal antibodies (mAbs) against the viral protein 1 (VP1) major capsid protein of BKV genotypes I-IV using VelocImmune(R) transgenic mice that encode for human immunoglobulins. Hybridoma clones from VelocImmune(R) mice immunized with combinations of BKV I-IV VP1 and respective genotypes of BKV pseudoviruses (BK-PsV) were screened using a high-throughput binding assay against BKV-I VP1 expressed in HEK-293 cells. The VP1-binding hybridoma clones were then assessed for neutralization with BK-PsV consisting of respective VP1 proteins from BKV-I, -II, -III, or -IV on the virion surface. Overall, thirty-six broadly cross-neutralizing mAbs against BKV-I, -II, -III, and -IV -were identified. Twenty of the cross-reactive immunoglobulins were subjected to nucleotide sequencing resulting in six clonotype families with fourteen genetically distinct immunoglobulins. Several of the most effective mAbs were fully humanized with the IgG1 Fc domain and broad neutralization of BK-PsV-I, II, III, and IV genotypes with IC50s ranging from [~]7-200 pM. Thus, these cross-neutralizing mAbs represent potential biologics to developed into BKV therapeutics.

microbiology↗

bioRxiv2024

De novo antibody discovery in human blood from full-length single B cell transcriptomics and matching haplotyped-resolved germline assemblies

Immunoglobulin (IGH, IGK, IGL) loci in the human genome are highly polymorphic regions that encode the building blocks of the light and heavy chain IG proteins that dimerize to form antibodies. The processes of V(D)J recombination and somatic hypermutation in B cells are responsible for creating an enormous reservoir of highly specific antibodies capable of binding a vast array of possible antigens. However, the antibody repertoire is fundamentally limited by the set of variable (V), diversity (D), and joining (J) alleles present in the germline IG loci. To better understand how the germline IG haplotypes contribute to the expressed antibody repertoire, we combined genome sequencing of the germline IG loci with single-cell transcriptome sequencing of B cells from the same donor. Sequencing and assembly of the germline IG loci captured the IGH locus in a single fully-phased contig where the maternal and paternal contributions to the germline V, D, and J repertoire can be fully resolved. The B cells were collected following a measles, mumps, and rubella (MMR) vaccination, resulting in a population of cells that were activated in response to this specific immune challenge. Single-cell, full-length transcriptome sequencing of these B cells resulted in whole transcriptome characterization of each cell, as well as highly-accurate consensus sequences for the somatically rearranged and hypermutated light and heavy chain IG transcripts. A subset of antibodies synthesized based on their consensus heavy and light chain transcript sequences demonstrated binding to measles antigens and neutralization of measles live virus.

immunology↗

bioRxiv2023

An in vitro experimental pipeline to characterize the binding specificity of SARS-CoV-2 neutralizing antibodies

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor binding domain (RBD) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferation in vivo (1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-EM. FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif (RBM). Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470-490 loop at the Spike RBD tip. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants. ImportanceThe COVID-19 pandemic remains a significant public health concern for the global population; development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as SARS-CoV-2 variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread with the caveat that they interact with the circulating variants. The epitope and binding specificity of a broadly neutralizing anti-SARS-CoV-2 Spike RBD antibody clone against many SARS-CoV-2 VOC was characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

microbiology↗

bioRxiv2022

Discovery of a SARS-CoV-2 Broadly-Acting Neutralizing Antibody with Activity against Omicron and Omicron + R346K Variants

The continual emergence of SARS-CoV-2 variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant, has rendered ineffective a number of previously EUA approved SARS-CoV-2 neutralizing antibody therapies. Furthermore, even those approved antibodies with neutralizing activity against Omicron are reportedly ineffective against the subset of Omicron variants that contain a R346K substitution, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. Following a campaign of antibody discovery based on the vaccination of Harbour H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of Spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against the Omicron and Omicron + R346K variants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for use in human clinical trials.

microbiology↗