Membrane-bound Antigen Research Articles

Multifunctional biomimetic nanosystem for retinoblastoma treatment

The nanodelivery carrier was modified with tumor cell membrane and dendritic cell membrane to create a double-membrane fusion biomimetic nanomedicine, termed FM@mPDA/DOX/JQ-1. This research introduces a biologically inspired platform for multimodal combined tumor therapy. Because fusion membrane nanomedicines inherit the homologous targeting properties of tumor cells, chemotherapy drugs can be delivered to tumor lesions. In addition, the fusion membrane carries abundant and complete tumor cell membrane antigens. Based on the professional antigen presentation characteristics of dendritic cells, it directly or indirectly promotes the uptake of cell membrane-bound tumor antigens and downstream immune responses. In ectopic and orthotopic tumor-bearing mouse models, FM@mPDA/DOX/JQ-1 can induce a durable immune response to inhibit the rebound of primary tumors after chemotherapy and photothermal therapy (PTT). FM@mPDA/DOX/JQ-1 demonstrates no adverse impact on liver and kidney function, as well as blood indicators in mice, affirming its excellent safety profile. This tumor-specific immunotherapy-based nanoplatform holds promise for extension across various tumor types, offering a viable approach for multimodal combined tumor therapy.

Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells.

Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in preclinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four-residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit can disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are powerful tools for expanding the CAR T cell engineering toolbox.

Generation and preclinical evaluation of a human heavy-chain-only antibody recognizing the membrane-bound tumor-associated antigen mesothelin

ObjectiveMesothelin (MSLN) is an attractive target for anticancer therapeutics and bioimaging reagents that utilize antibodies. This study was aimed at developing a novel human anti-MSLN single-domain antibody that exclusively binds to the membrane-attached MSLN using transgenic mice generating human heavy-chain-only antibodies (HCAbs) and exploring the resulting HCAbs as imaging tools.MethodsWe introduced a doxycycline-inducible human MSLN gene in genetically modified mice expressing human HCAbs. This new method of non-invasive immunization by antigen induction results in MSLN antigen production in its native conformation on the cell surface. Screening of 2,000 HCAbs from the resulting immune library yielded numerous binders, from which we chose 19G6 as the lead antibody. This antibody was 111Indium radiolabeled and tested in a xenotransplantation tumor model with OVCAR-3 cells.ResultsThe 19G6 antibody shows nanomolar affinity toward membrane-bound MSLN and does not recognize soluble MSLN. The human MSLN-positive tumors were visualized in an in vivo mouse model. The non-labeled antibody prevented binding when provided in excess, showing tumor specificity.Conclusion19G6 with a human Fc is a promising tumor-cell tracer in vivo. This HCAb can also be engineered into a smaller and shorter-lived tracer (only the VH domain) or combined with other target-binding domains to form multispecific modalities for tumor immunotherapy.

The ion channel TRPV5 regulates B-cell signaling and activation.

B-cell activation triggers the release of endoplasmic reticulum calcium stores through the store-operated calcium entry (SOCE) pathway resulting in calcium influx by calcium release-activated calcium (CRAC) channels on the plasma membrane. B-cell-specific murine knockouts of SOCE do not impact humoral immunity suggesting that alternative channels may be important. We identified a member of the calcium-permeable transient receptor potential (TRP) ion channel family, TRPV5, as a candidate channel expressed in B cells by a quantitative polymerase chain reaction (qPCR) screen. To further investigate the role of TRPV5 in B-cell responses, we generated a murine TRPV5 knockout (KO) by CRISPR-Cas9. We found TRPV5 polarized to B-cell receptor (BCR) clusters upon stimulation in a PI3K-RhoA-dependent manner. TRPV5 KO mice have normal B-cell development and mature B-cell numbers. Surprisingly, calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impaired; however, differential expression of other calcium-regulating proteins, such as ORAI1, may contribute to a compensatory mechanism for calcium signaling in these cells. We demonstrate that TRPV5 KO B cells have impaired spreading and contraction in response to membrane-bound antigen. Consistent with this, TRPV5 KO B cells have reduced BCR signaling measured through phospho-tyrosine residues. Lastly, we also found that TRPV5 is important for early T-dependent antigen specific responses post-immunization. Thus, our findings identify a role for TRPV5 in BCR signaling and B-cell activation.

Nirogacestat: First Approval.

Nirogacestat (OGSIVEO™) is an oral, selective, reversible, small molecule γ-secretase inhibitor developed by SpringWorks Therapeutics, Inc. γ-Secretase is a multi-subunit protease complex that cleaves multiple transmembrane protein complexes, including Notch and membrane-bound B-cell maturation antigen (BCMA). Inhibition of γ-secretase may result in growth inhibition of tumour cells overexpressing Notch, and preservation of membrane-bound BCMA may increase target density for BCMA-targeted therapy. In November 2023, nirogacestat was approved in the USA for use in adult patients with progressing desmoid tumours who require systemic treatment. This article summarizes the milestones in the development of nirogacestat leading to this first approval for the systemic treatment of desmoid tumours.

Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC50 in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.

Artificial Intelligence-Based Counting Algorithm Enables Accurate and Detailed Analysis of the Broad Spectrum of Spot Morphologies Observed in Antigen-Specific B-Cell ELISPOT and FluoroSpot Assays.

Antigen-specific B-cell ELISPOT and multicolor FluoroSpot assays, in which the membrane-bound antigen itself serves as the capture reagent for the antibodies that B cells secrete, inherently result in a broad range of spot sizes and intensities. The diversity of secretory footprint morphologies reflects the polyclonal nature of the antigen-specific B cell repertoire, with individual antibody-secreting B cells in the test sample differing in their affinity for the antigen, fine epitope specificity, and activation/secretion kinetics. To account for these heterogeneous spot morphologies, and to eliminate the need for setting up subjective counting parameters well-by-well, CTL introduces here its cutting-edge deep learning-based IntelliCount™ algorithm within the ImmunoSpot® Studio Software Suite, which integrates CTL's proprietary deep neural network. Here, we report detailed analyses of spots with a broad range of morphologies that were challenging to analyze using standard parameter-based counting approaches. IntelliCount™, especially in conjunction with high dynamic range (HDR) imaging, permits the extraction of accurate, high-content information of such spots, as required for assessing the affinity distribution of an antigen-specific memory B-cell repertoire ex vivo. IntelliCount™ also extends the range in which the number of antibody-secreting B cells plated and spots detected follow a linear function; that is, in which the frequencies of antigen-specific B cells can be accurately established. Introducing high-content analysis of secretory footprints in B-cell ELISPOT/FluoroSpot assays, therefore, fundamentally enhances the depth in which an antigen-specific B-cell repertoire can be studied using freshlyisolated or cryopreserved primary cell material, such as peripheral blood mononuclear cells.

Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting.

The enzyme-linked immunospot (ELISpot) assay is a highly useful and sensitive method to detect total immunoglobulin and antigen-specific antibody-secreting cells. In addition, this method can measure biological activity and immunological secretions from immune cells. In general, membrane-bound antigen allows binding of antibody secreted by B cells, or a membrane-bound analyte-specific antibody binds to the specific analyte (e.g., cytokines) elicited from cells added to the well containing the bound antibody. The response from added cells is then detected by using an anti-Ig antibody and a colorimetric substrate, while in the case of non-B cells, the elicited antigen is detected with appropriate antibodies and enzyme-conjugated antibodies. Specificity of antibodies binding the protein of interest is necessary to achieve correct results. Western blotting can be used for this with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. Despite its general simplicity, western blotting is a powerful technique for immunodetection of proteins (notably low abundance proteins) as it provides simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Now, we have plethora of immunoblotting methods to validate antibodies for ELISpot.

Prostate-Specific Membrane Antigen (PSMA)-Positive Extracellular Vesicles in Urine-A Potential Liquid Biopsy Strategy for Prostate Cancer Diagnosis?

Simple SummaryProstate cancer is the second most commonly diagnosed cancer and the fifth leading cause of cancer-related death in men. It is a generally slow-growing cancer that—when detected in its early stages—has high chances of successful treatment. Just like all cells, cancerously degenerated cells release extracellular vesicles (EVs) to communicate with other cells. The aim of our research was to specifically isolate prostate cancer-derived EVs from urine, characterize the EV surface markers of a prostate cancer cohort, and assess the potential value of the prostate-specific membrane antigen (PSMA) as a biomarker for liquid biopsy in early cancer diagnostics. Our findings demonstrate that the automated isolation of EVs allows for an overall improvement of the precision in sample purification in comparison to manual isolation, thus optimizing the further characterization of EV surface markers as well as evaluating their use in clinical application.All cells release extracellular vesicles (EVs) to communicate with adjacent and distant cells. Consequently, circulating EVs are found in all bodily fluids, providing information applicable for liquid biopsy in early cancer diagnosis. Studies observed an overexpression of the membrane-bound prostate-specific membrane antigen (PSMA) on prostate cancer cells. To investigate whether EVs derived from communicating prostate cells allow for reliable conclusions on prostate cancer development, we isolated PSMA-positive, as well as CD9-positive, EVs from cell-free urine with the use of magnetic beads. These populations of EVs were subsequently compared to CD9-positive EVs isolated from female urine in Western blotting, indicating the successful isolation of prostate-derived and ubiquitous EVs, respectively. Furthermore, we developed a device with an adapted protocol that enables an automated immunomagnetic enrichment of EVs of large sample volumes (up to 10 mL), while simultaneously reducing the overall bead loss and hands-on time. With an in-house spotted antibody microarray, we characterized PSMA as well as other EV surface markers of a prostate cohort of 44 urine samples in a more simplified way. In conclusion, the automated and specific enrichment of EVs from urine has a high potential for future diagnostic applications.

Strategies to Screen Anti-AQP4 Antibodies from Yeast Surface Display Libraries.

A rapid and effective method to identify disease-specific antibodies from clinical patients is important for understanding autoimmune diseases and for the development of effective disease therapies. In neuromyelitis optica (NMO), the identification of antibodies targeting the aquaporin-4 (AQP4) membrane protein traditionally involves the labor-intensive and time-consuming process of single B-cell sorting, followed by antibody cloning, expression, purification, and analysis for anti-AQP4 activity. To accelerate patient-specific antibody discovery, we compared two unique approaches for screening anti-AQP4 antibodies from yeast antibody surface display libraries. Our first approach, cell-based biopanning, has strong advantages for its cell-based display of native membrane-bound AQP4 antigens and is inexpensive and simple to perform. Our second approach, FACS screening using solubilized AQP4 antigens, permits real-time population analysis and precision sorting for specific antibody binding parameters. We found that both cell-based biopanning and FACS screening were effective for the enrichment of AQP4-binding clones. These screening techniques will enable library-scale functional interrogation of large natively paired antibody libraries for comprehensive analysis of anti-AQP4 antibodies in clinical samples and for robust therapeutic discovery campaigns.

Lipid Membrane-Based Antigen Presentation to B Cells Using a Fully Synthetic Ex Vivo Germinal Center Model.

High-affinity antigen-specific B cells are generated within specialized structures, germinal centers (GCs), inside lymphoid organs. In GCs, follicular dendritic cells (FDCs) present antigens on their membrane surface to cognate B cells, inducing rapid proliferation and differentiation of the B cells toward antibody-secreting cells. The FDC's fluid membrane surface allows B cells to "pull" the antigens into clusters and internalize them, a process that frequently involves tearing off and internalizing FDC membrane fragments. To study this process ex vivo, liposomal membranes are used as the antigen-presenting FDC-like fluid lipid surface to activate B cells. In a fully synthetic in vitro GC model (sGC), which uses the microbead-based presentation of the CD40 Ligand and a cytokine cocktail to mimic T follicular helper cell signals to B cells, liposomes presenting a model antigen mimic effectively engage B cell receptors (BCRs) and induce greater BCR clustering compared to soluble antigens, resulting in rapid antigen internalization and proliferation of the B cells. B cells showed GC-like reactions and undergo efficient IgG1 class-switching. Taken together, the results suggest that fluid membrane-bound antigen induces a strong GC response and provides a novel synthetic in vitro system for studying GC biology in health and diseases, and for expanding therapeutic B cells ex vivo.

A B-cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation.

B-cell activation and immune synapse (IS) formation with membrane-bound antigens are actin-dependent processes that scale positively with the strength of antigen-induced signals. Importantly, ligating the B-cell integrin, LFA-1, with ICAM-1 promotes IS formation when antigen is limiting. Whether the actin cytoskeleton plays a specific role in integrin-dependent IS formation is unknown. Here, we show using super-resolution imaging of mouse primary B cells that LFA-1:ICAM-1 interactions promote the formation of an actomyosin network that dominates the B-cell IS. This network is created by the formin mDia1, organized into concentric, contractile arcs by myosin 2A, and flows inward at the same rate as B-cell receptor (BCR):antigen clusters. Consistently, individual BCR microclusters are swept inward by individual actomyosin arcs. Under conditions where integrin is required for synapse formation, inhibiting myosin impairs synapse formation, as evidenced by reduced antigen centralization, diminished BCR signaling, and defective signaling protein distribution at the synapse. Together, these results argue that a contractile actomyosin arc network plays a key role in the mechanism by which LFA-1 co-stimulation promotes B-cell activation and IS formation.

Hybrid membrane-coated nanosuspensions for multi-modal anti-glioma therapy via drug and antigen delivery

BackgroundGlioma is one of the deadliest human cancers. Although many therapeutic strategies for glioma have been explored, these strategies are seldom used in the clinic. The challenges facing the treatment of glioma not only involve the development of chemotherapeutic drugs and immunotherapeutic agents, but also the lack of a powerful platform that could deliver these two moieties to the targeted sites. Herein, we developed chemoimmunotherapy delivery vehicles based on C6 cell membranes and DC membranes to create hybrid membrane-coated DTX nanosuspensions (DNS-[C6&DC]m).ResultsResults demonstrated successful hybrid membrane fusion and nanosuspension functionalization, and DNS-[C6&DC]m could be used for different modes of anti-glioma therapy. For drug delivery, membrane coating could be applied to target the source cancer cells via a homotypic-targeting mechanism of the C6 cell membrane. For cancer immunotherapy, biomimetic nanosuspension enabled an immune response based on the professional antigen-presenting characteristic of the dendritic cell membrane (DCm), which carry the full array of cancer cell membrane antigens and facilitate the uptake of membrane-bound tumor antigens for efficient presentation and downstream immune n.ConclusionDNS-[C6&DC]m is a multifunctional biomimetic nano-drug delivery system with the potential to treat gliomas through tumor-targeted drug delivery combined with immunotherapy, thereby presenting a promising approach that may be utilized for multiple modes of cancer therapy.Graphical

Marginal zone B cells have higher basal BCR signaling that correlates with altered mobility and spatial organization of IgM-BCRs

Abstract Marginal zone (MZ) B cells exist in a partially-activated ‘primed’ state but the molecular basis for this priming is not fully understood. We found that MZ B cells exhibit greater antigen-independent ‘tonic’ BCR signaling than naïve follicular (FO) B cells. Because BCR signaling output is dependent on BCR spatial organization and BCR-BCR interactions, we hypothesized that the increased tonic BCR signaling in MZ B cells is due to altered lateral mobility and nanoscale organization of BCRs. Single-particle tracking showed that surface IgM-BCRs on MZ B cells have higher diffusion coefficients and decreased confinement compared to IgM-BCRs on FO B cells. In contrast, the mobility and confinement of IgD-BCRs was similar on MZ and FO B cells. To assess BCR nanoscale organization, we used dSTORM and a novel graph-based clustering algorithm. This revealed that IgM-BCRs were more dispersed and less clustered on MZ B cells than on FO B cells, whereas IgD-BCR spatial organization was similar on the two cell populations. Importantly, 3-color STED imaging revealed that phospho-CD79 nanoclusters overlapped to a much greater extent with IgM-BCRs than with IgD-BCRs on FO B cells, and that this IgM-pCD79 overlap was even greater in MZ B cells. MZ B cells also exhibited greater pCD79 signaling in response to membrane-bound antigens than FO B cells. Thus, MZ B cells have greater tonic and antigen-dependent BCR signaling, which correlates with altered IgM-BCR mobility and organization

The Wdr1-LIMK-Cofilin Axis Controls B Cell Antigen Receptor-Induced Actin Remodeling and Signaling at the Immune Synapse.

When B cells encounter membrane-bound antigens, the formation and coalescence of B cell antigen receptor (BCR) microclusters amplifies BCR signaling. The ability of B cells to probe the surface of antigen-presenting cells (APCs) and respond to APC-bound antigens requires remodeling of the actin cytoskeleton. Initial BCR signaling stimulates actin-related protein (Arp) 2/3 complex-dependent actin polymerization, which drives B cell spreading as well as the centripetal movement and coalescence of BCR microclusters at the B cell-APC synapse. Sustained actin polymerization depends on concomitant actin filament depolymerization, which enables the recycling of actin monomers and Arp2/3 complexes. Cofilin-mediated severing of actin filaments is a rate-limiting step in the morphological changes that occur during immune synapse formation. Hence, regulators of cofilin activity such as WD repeat-containing protein 1 (Wdr1), LIM domain kinase (LIMK), and coactosin-like 1 (Cotl1) may also be essential for actin-dependent processes in B cells. Wdr1 enhances cofilin-mediated actin disassembly. Conversely, Cotl1 competes with cofilin for binding to actin and LIMK phosphorylates cofilin and prevents it from binding to actin filaments. We now show that Wdr1 and LIMK have distinct roles in BCR-induced assembly of the peripheral actin structures that drive B cell spreading, and that cofilin, Wdr1, and LIMK all contribute to the actin-dependent amplification of BCR signaling at the immune synapse. Depleting Cotl1 had no effect on these processes. Thus, the Wdr1-LIMK-cofilin axis is critical for BCR-induced actin remodeling and for B cell responses to APC-bound antigens.

Hydrophobicity and the Physico-Chemical Basis of Immunotolerance

This study analyzes the primary electrostatic interaction between the membrane-bound B-cell receptor and antigen peptide determinants, and identifies peptide frequency and hydrophobicity as the main factors that govern and shape immunotolerance versus immunoreactivity.

Pathogenic mechanisms of autoantibodies: antibodies to intracellular and membrane-bound antigens

Pathogenic mechanisms of autoantibodies: antibodies to intracellular and membrane-bound antigens

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100-1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450-900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.

Engineering Cellular Biosensors with Customizable Antiviral Responses Targeting Hepatitis B Virus.

SummarySynNotch receptor technology is a versatile tool that uses the regulatory notch core portion with an extracellular scFv and an intracellular transcription factor that enables to program customized input and output functions in mammalian cells. In this study, we designed a novel synNotch receptor comprising scFv against HBs antigen linked with an intracellular artificial transcription factor and exploited it for viral sensing and cellular immunotherapy. The synNotch receptor expressing cells sensed HBV particles and membrane-bound HBs antigens and responded by expressing reporter molecules, secNL or GFP. We also programmed these cells to dispense antiviral responses such as type I interferon and anti-HBV neutralizing mouse-human chimeric antibodies. Our data reveal that synNotch receptor signaling works for membrane-bound ligands such as enveloped viral particles and proteins borne on liposomal vesicles. This study establishes the concepts of “engineered immunity” where the synNotch platform is utilized for cellular immunotherapy against viral infections.

Teleost IgD+IgM− B Cells Mount Clonally Expanded and Mildly Mutated Intestinal IgD Responses in the Absence of Lymphoid Follicles

SummaryImmunoglobulin D (IgD) is an ancient antibody with dual membrane-bound and fluid-phase antigen receptor functions. The biology of secreted IgD remains elusive. Here, we demonstrate that teleost IgD+IgM− plasmablasts constitute a major lymphocyte population in some mucosal surfaces, including the gut mucosa. Remarkably, secreted IgD binds to gut commensal bacteria, which in turn stimulate IgD gene transcription in gut B cells. Accordingly, secreted IgD from gut as well as gill mucosae, but not the spleen, show a V(D)J gene configuration consistent with microbiota-driven clonal expansion and diversification, including mild somatic hypermutation. By showing that secreted IgD establishes a mutualistic relationship with commensals, our findings suggest that secreted IgD may play an evolutionary conserved role in mucosal homeostasis.