Biology subjects

Suh, S.

Publications and source records attributed to Suh, S..

7 recordsLinked to original sources

bioRxiv2025

An integrated in silico-in vitro workflow for discovering high-affinity, selective antibodies to the KRAS(G12D)-MHC I complex

Antibodies that recognize peptide-loaded class I major histocompatibility complex (pMHC I) molecules could enable therapeutic targeting of intracellular oncogenic proteins, yet their discovery has been hampered by the small size of peptide antigens and allele-specificity. We describe an integrated in silico-in vitro workflow for generating high-affinity, selective antibodies to KRAS(G12D)10 presented by HLA-C*08:02, a clinically validated cancer neoantigen. In silico, multiple human antibody-derived variable fragments (Fvs) plausibly docked to the target pMHC were generated, followed by limited complementarity-determining region (CDR) sequence design. In vitro, CDR diversity was introduced at 3-4 positions per Fv to construct yeast surface display library for iterative selections. This workflow yielded antibodies with exclusive binding to KRAS(G12D)10/HLA-C*08:02 without cross-reactivity. Affinity maturation achieved nanomolar dissociation constants, and incorporation into chimeric antigen receptor T cells enabled specific activation against target-positive cells. This study establishes a practical design-to-function pipeline for TCR-like antibody discovery, and demonstrates the feasibility of therapeutic targeting against KRAS(G12D)-driven malignancies.

biochemistry↗

bioRxiv2025

Computational design of a single-domain antibody that specifically recognizes WT1 peptide-loaded class I MHC

T cell receptor (TCR)-like antibodies that recognize peptide-loaded class I MHC (pMHC) complexes can enable precise targeting of cancer cells, but developing single-domain binders with high specificity and affinity is challenging. Here, we report the computational design and experimental validation of a TCR-like single-domain antibody (sdAb) that specifically recognizes the WT1-derived peptide RMFPNAPYL presented by HLA-A*02:01. Starting from the crystal structure of a Fab antibody bound to RMF/HLA- A02:01, we repurposed the VH domain into a stable, soluble Trastuzumab-derived VH scaffold. The resulting sdAb, RMFsdAb, spans all nine peptide residues and shows no binding to a control pMHC with a different peptide. Its biophysical properties were improved by fusion to human serum albumin domain III (HSA D3), yielding a monodisperse HSA D3-RMFsdAb with 81 nM affinity and specificity for RMF/HLA- A*02:01. We further engineered a bivalent format (RMFsdAb-HSA D3-RMFsdAb), which dramatically increased apparent binding affinity to 0.4 nM. When expressed on a CAR T cell, HSA D3-RMFsdAb functioned as the antigen-recognition domain to selectively activate T cells in the presence of RMF/HLA-A*02:01-positive cells. Our results demonstrate a viable strategy to develop high-specificity, peptide-focused TCR-like sdAbs for pMHC-targeted therapeutics.

biochemistry↗

bioRxiv2025

Computational design of monomeric Fc variants with distinct pH-responsive FcRn-binding profiles

IgG1 and IgG4 antibodies form a [~]150 kDa homodimer through dimerization of the Fc domain, which prolongs their in vivo half-life via pH-dependent binding to the neonatal Fc receptor (FcRn). Conformationally stable, half-life-extended monomeric Fc (mFc) variants offer a promising platform for antibodies and Fc-fusion therapeutics, enabling deeper tissue penetration, reduced toxicity, and simplified manufacturing. Due to the loss of binding avidity, mFc requires significantly enhanced FcRn-binding affinity at pH 6.0, but retaining weak binding at neutral pH to achieve comparable serum half-life, making engineering such mFc variants highly challenging. Mainly by computational design approach, we created mFc mutants with diverse human FcRn-binding profiles, including two variants that exhibit 17- and 47-fold stronger FcRn binding at pH 6.0 (KD of 30.7 nM and 88 nM) compared to a baseline mFc, while maintaining greater than 213-fold weaker binding at pH 7.4 (KD of 6,549 nM and 39,150 nM). These variants are highly soluble and display a melting temperature greater than 60.6 {degrees}C, underscoring their potential as platforms for extending the in vivo half-life of therapeutic modalities. Other mFc variants with different pH-responsive FcRn-binding profiles would potentially fit for other therapeutic needs. Moreover, transferring the same variations into IgG4 Fc generated IgG4 mFc variants with FcRn-binding properties similar to those of the parent IgG1 mFc variants. Furthermore, incorporating the FcRn-binding affinity-enhancing substitutions into native Fc produced a dimeric Fc variant that exhibits strong, pH-responsive FcRn-binding affinities, promising an extended half-life in serum.

bioengineering↗

bioRxiv2025

UHRF1 is critical for tumor-promoting inflammation and tumorigenesis in retinoblastoma

Retinoblastoma, the most common pediatric intraocular malignancy, arises from RB1 inactivation, leading to uncontrolled proliferation of retinal progenitor cells. Epigenetic dysregulation a key driver of retinoblastoma progression, yet the underlying mechanisms are poorly understood. UHRF1, a regulator of DNA methylation and chromatin remodeling, has been implicated in oncogenesis but its role in retinoblastoma has not been fully characterized. To investigate Uhrf1s role in tumor initiation and progression, we generated a genetically engineered mouse model of retinoblastoma with conditional Uhrf1 knockout. Remarkably, Uhrf1 loss completely blocked tumor formation, despite persistent early oncogenic events. Transcriptomic and epigenomic profiling revealed that Uhrf1 is essential for tumor progression, facilitating oncogenic transcription, chromatin accessibility, and aberrant DNA methylation. Beyond its epigenetic role, we found Uhrf1 modulates the tumor immune microenvironment, promoting chemokine secretion and microglial infiltration, suggesting that Uhrf1 promotes immune cell recruitment. Mechanistically, Uhrf1 enhances chemokine expression through NF-{kappa}B signaling, establishing a novel connection between epigenetic regulation and tumor-associated immune responses. These findings establish Uhrf1 as a critical driver of retinoblastoma progression, essential for tumor maintenance but not initiation. By sustaining oncogenic transcriptional programs and shaping a pro-tumor immune microenvironment, Uhrf1 emerges as a promising therapeutic target for inhibiting tumor growth and immune evasion.

cancer biology↗

bioRxiv2024

Single-cell and spatial transcriptomics of vulvar lichen sclerosus reveal multicompartmental alterations in gene expression and signaling cross-talk

Vulvar diseases are a critical yet often neglected area of womens health, profoundly affecting patients quality of life and frequently resulting in long-term physical and psychological challenges. Lichen sclerosus (LS) is a chronic inflammatory skin disorder that predominantly affects the vulva, leading to severe itching, pain, scarring, and an increased risk of malignancy. Despite its profound impact on affected individuals, the molecular pathogenesis of vulvar LS (VLS) is not well understood, hindering the development of FDA-approved therapies. Here, we utilize single-cell and spatial transcriptomics to analyze lesional and non-lesional skin from VLS patients, as well as healthy control vulvar skin. Our findings demonstrate histologic, cellular, and molecular heterogeneities within VLS, yet highlight unifying molecular changes across keratinocytes, fibroblasts, immune cells, and melanocytes in lesional skin. They reveal cellular stress and damage in fibroblasts and keratinocytes, enhanced T cell activation and cytotoxicity, aberrant cell-cell signaling, and increased activation of the IFN, JAK/STAT, and p53 pathways in specific cell types. Using both monolayer and organotypic culture models, we also demonstrate that knockdown of select genes, which are downregulated in VLS lesional keratinocytes, partially recapitulates VLS-like stress-associated changes. Collectively, these data provide novel insights into the pathogenesis of VLS, identifying potential biomarkers and therapeutic targets for future research.

genomics↗

bioRxiv2021

Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to co-immunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each samples copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which were enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition provides a model for future studies of other complex cellular structures, and gives new insights into how intricate membrane structure and protein activity are balanced within the ROS. SUMMARYSander et al. have parsed the lipid composition of native-source photoreceptor disks and find large differences in fatty acid unsaturation and chain length between the center and rim regions. They selectively copurify membrane proteins and lipids from each region in SMALPs using nanobodies and antibodies.

cell biology↗

bioRxiv2020

Long-term cellulose enrichment selects for highly cellulolytic consortia and competition for public goods

The complexity of microbial communities hinders our understanding of how microbial diversity and microbe-microbe interactions impact community functions. Here, using six independent communities originating from the refuse dumps of leaf-cutter ants and enriched using the plant polymer cellulose as the sole source of carbon, we examine how changes in bacterial diversity and interactions impact plant biomass decomposition. Over up to 60 serial transfers ([~]8 months), cellulolytic ability increased then stabilized in four enrichment lines and was variable in two lines. Bacterial community characterization using 16S rRNA gene amplicon sequencing showed community succession differed between the highly cellulolytic and variably cellulolytic enrichment lines. Metagenomic and metatranscriptomic analyses revealed that Cellvibrio and/or Cellulomonas dominated each enrichment line and produced the majority of cellulase enzymes, while diverse taxa were retained within these communities over the duration of transfers. Interestingly, the less cellulolytic communities had a higher diversity of organisms competing for the cellulose breakdown product cellobiose, suggesting that cheating slowed cellulose degradation. In addition, we found competitive exclusion as an important factor shaping all the communities, with the mutual exclusion of specific cellulolytic taxa within individual enrichment lines and the high expression of genes associated with the production of antagonistic compounds. Our results provide insights into how microbial diversity and competition affect the stability and function of cellulose-degrading communities. ImportanceMicrobial communities are a key driver of the carbon cycle through the breakdown of complex polysaccharides in diverse environments including soil, marine systems, and the mammalian gut.However, due to the complexity of these communities, the species-species interactions that impact community structure and ultimately shape the rate of decomposition are difficult to define. Here we performed serial enrichment on cellulose using communities inoculated from leaf-cutter ant refuse dumps, a cellulose-rich environment. By concurrently tracking cellulolytic ability and community composition and through metagenomic and metatranscriptomic sequencing, we analyzed the ecological dynamics of the enrichment lines. Our data suggest that antagonism is prevalent in these communities and that competition for soluble sugars may slow degradation and lead to community instability. Together, these results help reveal the relationships between competition and polysaccharide decomposition, with implications in diverse areas ranging from microbial community ecology to cellulosic biofuels production.

microbiology↗