Selection Of Antibodies Research Articles

Obicetrapib Treatment Increases Pre-Beta1 HDL and Lipophilic Antioxidants in the Ocean and Rose2 Studies

Background and Aims: Non-lipidated ApoA1 (pre-beta1 HDL) captures cholesterol and lipophilic antioxidants via ABCA1. HDL particles transport antioxidants to tissues such as retina and brain. The effect of HDL raising interventions on pre-beta1 HDL levels and the distribution of lipophilic antioxidants amongst lipoproteins is unknown. CETP-inhibition was hypothesized to reduce the antioxidant capacity of HDL, although direct evidence was never presented. This is now addressed in phase II studies of the novel CETP-inhibitor obicetrapibMethods: Plasma samples from two phase II studies with obicetrapib were used to measure changes in pre-beta1 HDL by ELISA with a selective antibody to ApoA1 aa 137-144 (LQEKLSPL).. Lipophilic antioxidants were quantified in plasma, non-HDL, and HDL-fractions by ULPC ms-ms.Results: In both studies, no significant changes occurred in placebo groups but, in the obicetrapib treated groups, plasma pre-beta1 HDL increased by 12% (p<0.04) and 24% (p<0.03). In OCEAN, plasma ApoA1 and HDL-C levels correlated with increasing levels of antioxidants in the HDL fraction.In ROSE2, α-tocopherol was raised in plasma by 16% (p<0.003) and in HDL by 58% (p<0.00003). In both studies pre-beta1 HDL in obicetrapib treated patients was correlated with α-tocopherol in plasma.Conclusions: CETP inhibition with obicetrapib increases pre-beta1 HDL, impacts excess cholesterol efflux, and also raises important HDL antioxidants. Thus, these results support the potential therapeutic use of obicetrapib in diseases with high unmet medical need that are associated with low HDL and low levels of lipophilic antioxidants in plasma and tissues, such as AMD, neurodegenerative disorders, and sickle cell anemia.

Igome Graphs Suggest the Changes in the IgM and IgG Repertoires in Antiphospholipid Syndrome Are in Part Idiotypically Defined

Antiphospholipid Syndrome (APS) is characterized by the persistence of high-affinity antiphospholipid antibodies, manifesting clinically as pregnancy complications, thrombosis, or neurological symptoms. This study explores the changes in the IgM and IgG antibody repertoires in APS patients using igome graphs, which represent antibody repertoires through peptide libraries selected from phage display on the complete immunoglobulin fraction of the serum. Serum samples from healthy women and APS patients were processed to isolate IgM and IgG fractions, which were then used for phage selection and next-generation sequencing. Peptide sequences were analyzed using graph representation, clustering, and spectral embedding to identify significant differences between the repertoires. The study revealed that while IgM repertoires in APS patients exhibit a loss of public reactivities, the IgG repertoires do not follow this trend. Three orthogonal sequence motifs were identified in the IgM and IgG repertoires, suggesting potential idiotypic interactions. These findings highlight the intricate mechanisms of antibody selection and cross-reactivity in APS, offering insights into potential therapeutic approaches. The study underscores the importance of system-level analysis in understanding immune mechanisms and suggests that idiotypic interactions, though complex, could play a crucial role in APS pathogenesis.

Selection and Characterization of Antibodies Recognizing Unnatural Base Pairs

Selection and Characterization of Antibodies Recognizing Unnatural Base Pairs

Selection of LRP1 ligand phage-displayed single domain antibody that transmigrates BBB

Effective drug delivery to the central nervous system (CNS) remains a challenge due to the blood–brain barrier (BBB). Macromolecules such as proteins and peptides are unable to cross BBB and have poor therapeutic efficacy due to little or no drug distribution. A promising alternative is the conjugation of a drug to a shuttle molecule that can reach the CNS via receptor-mediated transcytosis (RMT). Several receptors have been described for RMT, such as low-density lipoprotein receptor-related protein 1 (LRP1). We used phage display technology combined with an in vitro BBB model to identify LRP1 ligands. A single domain antibody (dAb) library was used to enrich for species that selectively bind to immobilised LRP1 ligand. We obtained a novel nanobody, dAb D11, that selectively binds to LRP1 receptor and mediates in vitro internalisation of phage particles in brain endothelial cells, with a dissociation constant Kd of 183.1 ± 85.8 nM. The high permeability of D11 was demonstrated by an in vivo biodistribution assay in mice. We discovered D11, the first LRP1 binding dAb with BBB permeability. Our findings will contribute to the development of RMT-based drugs for the treatment of CNS diseases.

Antibody selection and automated quantification of TRPV1 immunofluorescence on human skin

Assessing localization of the transient receptor potential vanilloid-1 (TRPV1) in skin nerve fibers is crucial for understanding its role in peripheral neuropathy and pain. However, information on the specificity and sensitivity of TRPV1 antibodies used for immunofluorescence (IF) on human skin is currently lacking. To find a reliable TRPV1 antibody and IF protocol, we explored antibody candidates from different manufacturers, used rat DRG sections and human skin samples for screening and human TRPV1-expressing HEK293 cells for further validation. Final specificity assessment was done on human skin samples. Additionally, we developed two automated image analysis methods: a Python-based deep-learning approach and a Fiji-based machine-learning approach. These methods involve training a model or classifier for nerve fibers based on pre-annotations and utilize a nerve fiber mask to filter and count TRPV1 immunoreactive puncta and TRPV1 fluorescence intensity on nerve fibers. Both automated analysis methods effectively distinguished TRPV1 signals on nerve fibers from those in keratinocytes, demonstrating high reliability as evidenced by excellent intraclass correlation coefficient (ICC) values exceeding 0.75. This method holds the potential to uncover alterations in TRPV1 associated with neuropathic pain conditions, using a minimally invasive approach.

Notch1 blockade by a novel, selective anti-Notch1 neutralizing antibody improves immunotherapy efficacy in melanoma by promoting an inflamed TME

BackgroundImmune checkpoint inhibitors (ICI) have dramatically improved the life expectancy of patients with metastatic melanoma. However, about half of the patient population still present resistance to these treatments. We have previously shown Notch1 contributes to a non-inflamed TME in melanoma that reduces the response to ICI. Here, we addressed the therapeutic effects of a novel anti-Notch1 neutralizing antibody we produced, alone and in combination with immune checkpoint inhibition in melanoma models.MethodsAnti-Notch1 was designed to interfere with ligand binding. Mice were immunized with a peptide encompassing EGF-like repeats 11–15 of human Notch1, the minimal required region that allows ligand binding and Notch1 activation. Positive clones were expanded and tested for neutralizing capabilities. Anti-Notch1-NIC was used to determine whether anti-Notch1 was able to reduce Notch1 cleavage; while anti-SNAP23 and BCAT2 were used as downstream Notch1 and Notch2 targets, respectively. K457 human melanoma cells and the YUMM2.1 and 1.7 syngeneic mouse melanoma cells were used. Cell death after anti-Notch1 treatment was determined by trypan blue staining and compared to the effects of the gamma-secretase inhibitor DBZ. 10 mg/kg anti-Notch1 was used for in vivo tumor growth of YUMM2.1 and 1.7 cells. Tumors were measured and processed for flow cytometry using antibodies against major immune cell populations.ResultsAnti-Notch1 selectively inhibited Notch1 but not Notch2; caused significant melanoma cell death in vitro but did not affect normal melanocytes. In vivo, it delayed tumor growth without evident signs of gastro-intestinal toxicities; and importantly promoted an inflamed TME by increasing the cytotoxic CD8+ T cells while reducing the tolerogenic Tregs and MDSCs, resulting in enhanced efficacy of anti-PD-1.ConclusionsAnti-Notch1 safely exerts anti-melanoma effects and improves immune checkpoint inhibitor efficacy. Thus, anti-Notch1 could represent a novel addition to the immunotherapy repertoire for melanoma.

Unveiling the structural mechanisms behind high affinity and selectivity in phosphorylated epitope-specific rabbit antibodies

Protein phosphorylation is a crucial process in various cellular functions, and its irregularities have been implicated in several diseases, including cancer. Antibodies are commonly employed to detect protein phosphorylation in research. However, unlike the extensive studies on recognition mechanisms of the phosphate group by proteins such as kinases and phosphatases, only a few studies have explored antibody mechanisms. In this study, we produced and characterized two rabbit monoclonal antibodies that recognize a mono-phosphorylated Akt peptide. Through crystallography, thermodynamic mutational analyses, and molecular dynamics simulations, we investigated the unique recognition mechanism that enables higher binding affinity and selectivity of the antibodies compared to other generic proteins with lower binding affinity to phosphorylated epitopes. Our results demonstrate that molecular dynamics simulations provide novel insights into the dynamic aspects of molecular recognition of post-translational modifications by proteins beyond static crystal structures, highlighting how specific atomic level interactions drive the exceptional affinity and selectivity of antibodies.

A hapten design strategy to enhance the selectivity of monoclonal antibodies against malachite green

Malachite green (MG), a triphenylmethane dye, is used in the aquaculture industry as a disinfectant and insect repellent due to its potent bactericidal and pesticidal properties. However, its use poses potential environmental and health risks. This study analyzed and designed two haptens using computer simulation. Serum data confirmed the feasibility of introducing an arm at the dimethylamine group. Subsequently, a highly selective monoclonal antibody strain was successfully prepared based on the hapten. After optimizing the working conditions of indirect competitive enzyme-linked immunosorbent assay (IC-ELISA), the IC50 value was 0.83 ng/mL, with a detection limit (IC10) of 0.08 ng/mL and a linear range of 0.19–3.52 ng/mL. The developed monoclonal antibody exhibited a crossover rate of less than 0.1% with other similar structures and can be used to establish an immunoassay.

B-245 Novel monoclonal antibodies validated specifically for immunohistochemistry by performance assessment on tissue microarrays

Abstract Background Immunohistochemistry (IHC) is a laboratory immunoassay that is a pillar of diagnostic pathology, particularly in the identification and characterization of neoplasms and other diseases/disorders. Though performed routinely, rigorous SOP protocols must be determined empirically for each molecular target, beginning with antibody selection. Both traditional polyclonal and hybridoma-based monoclonal antibodies have been employed in the past, though monoclonal, and particularly recombinant monoclonal, antibodies are preferred. However, any reagent used for diagnostic purposes must be meticulously validated to minimize, if not eliminate, erroneous interpretations of IHC assays. Thus, the extent of validation is the pivotal aspect for establishing an antibody as a reliable diagnostic tool for IHC applications. Methods GeneTex has assembled an extensive catalog of rabbit and mouse monoclonal antibodies validated specifically for IHC. These antibodies are tested on both human normal tissue and cancer tissue microarrays, with upwards of 76 normal and numerous tumor tissues included. Images of the staining are available for all the tested tissues. This broad spectrum of tissues allows a researcher to assess the performance of the antibody on known positive and negative samples, thereby minimizing the risk of false positive or false negative readouts (Gown, 2016). In addition, the data images are of sufficient resolution to determine proper cellular localization of the signal. This special catalog of IHC-optimized reagents are listed under the “HistoMAX” designation. New antibodies will be added continuously, many of them being fully recombinant rabbit monoclonals manufactured using a multi-parameter FACS-based methodology to select antigen-specific IgG+ memory B cells from an immunized rabbit with subsequent cloning of the matched IgG heavy and light chains (Starkie et al., 2016). These recombinant antibodies will be validated following the same approach described above. Results Presently more than 130 mouse or rabbit monoclonal antibodies are available in the HistoMAX line of IHC-validated products and are directed against a spectrum of important targets. This includes antibodies for the commonly analyzed cytokeratins 5, 6, 7, 18, 19, and 20, among many others. Antibodies for CTLA4 and PD-L1 headline products for immunology and immunotherapy research, while other reagents are directed against various cadherins. Hormone receptor antibodies are also represented, with antibodies against estrogen receptor alpha, progesterone receptor, Her2/ErbB2, and the androgen receptor. There are also antibodies against various key “Cluster of Differentiation” (CD) proteins as well as other membrane proteins such as ACE2. Many antibodies are relevant to the study of human malignancies, such as CD66e/CEACAM5/CEA, MSH6, p63, MUC1, Collagen IV, EpCAM, PAX6, PAX8, GP2, TRIM29, S100, and GAD65. All antibodies were evaluated as described above. Conclusions Antibodies are application-specific reagents, and the best antibodies for IHC are validated through systematic staining of a multitude of normal and abnormal tissues. This is the only way that an antibody’s performance for IHC can be critically assessed. The HistoMAX reagents consist only of those monoclonal antibodies that have generated expected signals, or the lack of a signal, in a wide range of tissues in a manner consistent with the literature.

B-246 Pab to mab conversion via mass spectrometry: direct antibody discovery from serum

Abstract Background Navigating the intricacies of discovering unique and functional antibody binders for in vitro diagnostic development comes with several technological challenges, such as limited diversity of antibody repertoires, functional screening challenges, and the absence of suitable in vitro models. A proteomics-driven strategy to antibody discovery is a promising solution to overcome these obstacles by facilitating polyclonal to monoclonal antibody conversion. De novo polyclonal antibody sequencing utilizes mass spectrometry and machine-learning driven bioinformatics to obtain complete antibody sequences from immunoserum or purified protein samples. This approach investigates the entire circulating antibody repertoire at the protein level, thereby broadening the spectrum of potential candidates. It further facilitates functional selection by enabling antibody purification and enrichment techniques. This capability facilitates the identification of antibodies possessing unique functions, crucial for subsequent downstream development. We demonstrate the application and de novo polyclonal antibody sequencing using mass spectrometry and machine learning bioinformatics to enable the next generation of antibody discovery. Methods In this study, we employed a novel mass spectrometry-based approach, REpAb, to de novo sequence polyclonal antibodies directly from the immunized serum of animals or human patients. REpAb integrates mass spectrometry with machine learning-based bioinformatics to de novo sequence and assemble monoclonal antibodies from a polyclonal mixture. Protein lysates underwent analysis using an Orbitrap EclipseTM Series instrument (ThermoFisher Scientific, CA, US) coupled with the LC Evosep One (Evosep, Denmark). Results De novo polyclonal sequencing with REpAb yielded unique antibody binders not found in splenocytes or peripheral blood mononuclear cells (PBMCs). De novo antibody sequencing-discovered antibody sequences from naturally exposed humans showed unrestricted germline usage, compared to restricted germline used in antigen-immunized animals. Additionally, a proteomics-based approach for antibody discovery enables functional antibody selection through standard protein A/G purification, followed by negative and positive selection enrichment strategies to isolate antibody clones specific to the region of interest on the antigen. Conclusions Sequencing antibodies at the protein level directly reflects the circulating antibody repertoires, which expands the antibody diversity from the BCR repertoire. Proteomic-based antibody discovery empowers the functional selection of antibodies via meticulously designed enrichment strategies. This approach allows for the identification of a diverse pool of candidates possessing specific functions, such as anti-idiotypic features.

A guide to selecting high-performing antibodies for Rab10 (UniProt ID: P61026) for use in western blot, immunoprecipitation, and immunofluorescence

Rab10 is a small GTPase involved in cargo transport from the trans-Golgi network to the plasma membrane and endocytic recycling back to the cell membrane. It has garnered significant interest in neurodegenerative disease research, particularly due to its phosphorylation by the LRRK2 kinase. This relationship underscores the importance of Rab10 in cellular processes related to disease pathology, specifically Parkinson’s disease. The accessibility of renewable and selective antibodies against Rab10 would advance research efforts, enabling further understanding of its function and implications in disease. Here, we have characterized eight Rab10 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Advancing SARS‐CoV‐2 Variant Detection with High Affinity Monoclonal Antibodies and Plasmonic Nanostructure

AbstractA nanoplasmonic biosensor for the precise detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by leveraging advances in nanostructured sensing materials and highly selective monoclonal antibodies is presented. The sensor integrates plasmonic Au nanostructures optimized for surface‐enhanced Raman scattering (SERS), enhancing sensitivity through unique light interactions at the nanoscale. Coupled with exclusive antibodies that specifically target SARS‐CoV‐2 and its evolving variants, this sensor demonstrates remarkable selectivity and versatility. Validated with 270 clinical samples, it demonstrates a sensitivity of 98.9% and specificity of 100%. More importantly, the virus in nasopharyngeal swab samples collected over 3 years, marking the long‐term, large‐scale clinical validation of the nanoplasmonic biosensor for SARS‐CoV‐2 is been successfully detected. Furthermore, the integration of this sensor with face masks enables the detection of airborne SARS‐CoV‐2, highlighting its potential for routine management of coronavirus disease 2019 (COVID‐19).

Generation of Chicken Antibody Libraries and Selection of Antigen Binders.

Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.

Discovering Dengue Virus Antibody Using Phage Display Technology

Dengue virus (DENV) is a type of flavivirus transmitted globally by the Aedes aegypti mosquito and is the causal agent of Dengue fever. Due to global warm-ing and rising temperatures worldwide, the rate of exposure to DENV is now reaching unprecedented levels, with over 400 million people getting infected each year. Despite worldwide attention to DENV, there is not currently an effective treatment or vaccine. Here, we focused on developing an antibody for the NS1 protein of the DENV, which enables earlier detection of disease and could further decrease the rate of death. Our study utilized a method more time-saving and cost-effective than the traditional method of extracting antibodies from the blood or B-cells of infected animals that show immu-nity: Phage Display. This technique allowed us to derive monoclonal antibodies (mAbs) from the genome library and select DENV antibodies that bind to antigen proteins. In this study, we used Phage Display to select ninety-six potential antibodies from three phases of panning, which then went through indirect ELISA screening for optical density (OD) confirmation (OD &gt; 1.5). Sixteen of the high optical density antibodies were sent to a DNA sequencing analysis facility where fifteen of them were revealed to be the same 1G1 strain. By understanding how antibodies are discovered using phage display technology, we hope to advance the field with reevaluation of current vaccines and the development of novel ones.

An Engineered N-Glycosylated Dengue Envelope Protein Domain III Facilitates Epitope-Directed Selection of Potently Neutralizing and Minimally Enhancing Antibodies.

The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed "EDIII mutant N". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.

Intranuclear Irradiation Inhibits Solid Tumor Growth by Upregulating Caspase8 and Activating Apoptosis.

Radioimmunotherapy (RIT) is a novel and promising cancer treatment method, with ongoing research focusing on RIT antibody selection, radionuclides, treatment options, and benefited patient groups. As we dive into the mechanisms of tumor biology, a deeper exploration of how RIT affects tumor tissue is needed to provide new ways to improve clinical treatment outcome and patient prognosis. We labeled the anti-PD-L1 monoclonal antibody atezolizumab with iodine-131 (131I), separated and purified the labeled mAb with Sephadex G-25 medium gel filtration resin, and tested product stability. We detected the in vivo activity of 131I-PD-L1 mAb by analyzing its in vivo biodistribution and performing SPECT imaging and then set different treatment groups to study the effect of 131I-atezolizumab on the survival of tumor-bearing mice. Western blot, real-time quantitative PCR, and immunohistochemistry were used to detect the expression level of Caspase8 and Nlrp3 in tumor. TUNEL fluorescence staining was used to detect the apoptosis in the tumor. The radiopharmaceutical molecular probe 131I-atezolizumab showed high stability and in vivo biological activity. The treatment regimen adopted had a positive effect on the survival of tumor-bearing mice. 131I internal irradiation upregulated Caspase8 in tumor and ultimately inhibited solid tumor growth by activating apoptosis pathways. We also found a significant increase in the expression of NLRP3, which plays an important role in the pyroptosis pathway, in tumor. In summary, our data demonstrated that radiopharmaceuticals combined with immunotherapy affected tumor tissue by modulating relevant biological pathways, thereby achieving better antitumor effects compared with single therapy and providing new insights for promoting better patient prognosis and combination treatment strategies.

Heterologous Prime-Boost with Immunologically Orthogonal Protein Nanoparticles for Peptide Immunofocusing.

Protein nanoparticles are effective platforms for antigen presentation and targeting effector immune cells in vaccine development. Encapsulins are a class of protein-based microbial nanocompartments that self-assemble into icosahedral structures with external diameters ranging from 24 to 42 nm. Encapsulins from Myxococcus xanthus were designed to package bacterial RNA when produced in E. coli and were shown to have immunogenic and self-adjuvanting properties enhanced by this RNA. We genetically incorporated a 20-mer peptide derived from a mutant strain of the SARS-CoV-2 receptor binding domain (RBD) into the encapsulin protomeric coat protein for presentation on the exterior surface of the particle, inducing the formation of several nonicosahedral structures that were characterized by cryogenic electron microscopy. This immunogen elicited conformationally relevant humoral responses to the SARS-CoV-2 RBD. Immunological recognition was enhanced when the same peptide was presented in a heterologous prime/boost vaccination strategy using the engineered encapsulin and a previously reported variant of the PP7 virus-like particle, leading to the development of a selective antibody response against a SARS-CoV-2 RBD point mutant. While generating epitope-focused antibody responses is an interplay between inherent vaccine properties and B/T cells, here we demonstrate the use of orthogonal nanoparticles to fine-tune the control of epitope focusing.

Rapid Screening of Emergent Febuxostat Adulteration in Functional Foods Based on a Simulation-Inspired High-Quality Antibody.

Due to the potential health risks of adulterated febuxostat in uric-acid-lowering foods, it is urgent to develop rapid detection methods. However, there are no fast analytical techniques for febuxostat yet. Herein, an efficient hapten simulation strategy was proposed to successfully produce a highly sensitive and selective monoclonal antibody toward febuxostat. Based on such a robust recognition element, easy colorimetric and ultrasensitive fluorescent lateral flow immunochromatographic immunoassays were first established, which can detect febuxostat as low as 60 μg/kg by the naked eye or 1.01 μg/kg by a commercial test strip reader with acceptable stability. Furthermore, in the recovery test and blind sample analysis, consistent results between our methods and the authorized liquid chromatography-tandem mass spectrometry method suggested the high accuracy and practicality of this work. The present work not only proposes a rational hapten design idea but also provides favorable tools for the rapid screening of febuxostat in functional foods.

Two-dimensional high-throughput on-cell screening of immunoglobulins against broad antigen repertoires

Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.

An antibody that inhibits TGF-β1 release from latent extracellular matrix complexes attenuates the progression of renal fibrosis.

Inhibitors of the transforming growth factor-β (TGF-β) pathway are potentially promising antifibrotic therapies, but nonselective simultaneous inhibition of all three TGF-β homologs has safety liabilities. TGF-β1 is noncovalently bound to a latency-associated peptide that is, in turn, covalently bound to different presenting molecules within large latent complexes. The latent TGF-β-binding proteins (LTBPs) present TGF-β1 in the extracellular matrix, and TGF-β1 is presented on immune cells by two transmembrane proteins, glycoprotein A repetitions predominant (GARP) and leucine-rich repeat protein 33 (LRRC33). Here, we describe LTBP-49247, an antibody that selectively bound to and inhibited the activation of TGF-β1 presented by LTBPs but did not bind to TGF-β1 presented by GARP or LRRC33. Structural studies demonstrated that LTBP-49247 recognized an epitope on LTBP-presented TGF-β1 that is not accessible on GARP- or LRRC33-presented TGF-β1, explaining the antibody's selectivity for LTBP-complexed TGF-β1. In two rodent models of kidney fibrosis of different etiologies, LTBP-49247 attenuated fibrotic progression, indicating the central role of LTBP-presented TGF-β1 in renal fibrosis. In mice, LTBP-49247 did not have the toxic effects associated with less selective TGF-β inhibitors. These results establish the feasibility of selectively targeting LTBP-bound TGF-β1 as an approach for treating fibrosis.