Chromatin Immunoprecipitation of Retroviral Genomes with Antibodies Recognizing Modified Histones and Specific Viral Proteins.

Mammalian cells have developed and optimized defense mechanisms to prevent or hamper viral infection. The early transcriptional silencing of incoming viral DNAs is one such antiviral strategy and seems to be of fundamental importance, since most cell types silence unintegrated retroviral DNAs. In this chapter, a method for chromatin immunoprecipitation of unintegrated DNA is described. This technique allows investigators to examine histone and co-factor interactions with unintegrated viral DNAs as well as to analyze histone modifications in general or in a kinetic fashion at various time points during viral infection.

Investigating surface proteins and antibody combinations for detecting circulating tumor cells of various sarcomas.

Circulating tumor cells (CTCs) have gathered attention as a biomarker for carcinomas. However, CTCs in sarcomas have received little attention. In this work, we investigated cell surface proteins and antibody combinations for immunofluorescence detection of sarcoma CTCs. A microfluidic device that combines filtration and immunoaffinity using gangliosides 2 and cell surface vimentin (CSV) antibodies was employed to capture CTCs. For CTC detection, antibodies against cytokeratins 7 and 8 (CK), pan-cytokeratin (panCK), or a combination of panCK and CSV were used. Thirty-nine blood samples were collected from 21 patients of various sarcoma subtypes. In the independent samples study, samples were subjected to one of three antibody combination choices. Significant difference in CTC enumeration was found between CK and panCK + CSV, and between panCK and panCK + CSV. Upon stratification of CK+ samples, those of metastatic disease had a higher CTC number than those of localized disease. In the paired samples study involving cytokeratin-positive sarcoma subtypes, using panCK antibody detected more CTCs than CK. Similarly, for osteosarcoma, using panCK + CSV combination resulted in a higher CTC count than panCK. This study emphasized deliberate selection of cell surface proteins for sarcoma CTC detection and subtype stratification for studying cancers as heterogeneous as sarcomas.

Epitope binning for multiple antibodies simultaneously using mammalian cell display and DNA sequencing.

Epitope binning, an approach for grouping antibodies based on epitope similarities, is a critical step in antibody drug discovery. However, conventional methods are complex, involving individual antibody production. Here, we established Epitope Binning-seq, an epitope binning platform for simultaneously analyzing multiple antibodies. In this system, epitope similarity between the query antibodies (qAbs) displayed on antigen-expressing cells and a fluorescently labeled reference antibody (rAb) targeting a desired epitope is analyzed by flow cytometry. The qAbs with epitope similar to the rAb can be identified by next-generation sequencing analysis of fluorescence-negative cells. Sensitivity and reliability of this system are confirmed using rAbs, pertuzumab and trastuzumab, which target human epidermal growth factor receptor 2. Epitope Binning-seq enables simultaneous epitope evaluation of 14 qAbs at various abundances in libraries, grouping them into respective epitope bins. This versatile platform is applicable to diverse antibodies and antigens, potentially expediting the identification of clinically useful antibodies.

Electrically Oriented Antibodies on Transistor for Monitoring Several Copies of Methylated DNA.

An antibody transistor is a promising biosensing platform for the diagnosis and monitoring of various diseases. Nevertheless, the low concentration and short half-life of biomarkers require biodetection at the trace-molecule level, which remains a challenge for existing antibody transistors. Herein, we demonstrate a graphene field-effect transistor (gFET) with electrically oriented antibody probes (EOA-gFET) for monitoring several copies of methylated DNA. The electric field confines the orientation of antibody probes on graphene and diminishes the distance between graphene and methylated DNAs captured by antibodies, generating more induced charges on graphene and amplifying the electric signal. EOA-gFET realizes a limit of detection (LoD) of ∼0.12 copy µL-1, reaching the lowest LoD reported before. EOA-gFET shows a distinguishable signal for liver cancer clinical serum samples within ∼6 min, which proves its potential as a powerful tool for disease screening and diagnosis.

Understanding the Specific Implications of Amino Acids in the Antibody Development.

As the demand for immunotherapy to treat and manage cancers, infectious diseases and other disorders grows, a comprehensive understanding of amino acids and their intricate role in antibody engineering has become a prime requirement. Naturally produced antibodies may not have the most suitable amino acids at the complementarity determining regions (CDR) and framework regions, for therapeutic purposes. Therefore, to enhance the binding affinity and therapeutic properties of an antibody, the specific impact of certain amino acids on the antibody's architecture must be thoroughly studied. In antibody engineering, it is crucial to identify the key amino acid residues that significantly contribute to improving antibody properties. Therapeutic antibodies with higher binding affinity and improved functionality can be achieved through modifications or substitutions with highly suitable amino acid residues. Here, we have indicated the frequency of amino acids and their association with the binding free energy in CDRs. The review also analyzes the experimental outcome of two studies that reveal the frequency of amino acids in CDRs and provides their significant correlation between the outcomes. Additionally, it discusses the various bond interactions within the antibody structure and antigen binding. A detailed understanding of these amino acid properties should assist in the analysis of antibody sequences and structures needed for designing and enhancing the overall performance of therapeutic antibodies.

Unleashing the power of antibodies: Engineering for tomorrow's therapy.

Antibodies play a crucial role in host defense against various diseases. Antibody engineering is a multidisciplinary field that seeks to improve the quality of life of humans. In the context of disease, antibodies are highly specialized proteins that form a critical line of defense against pathogens and the disease caused by them. These infections trigger the innate arm of immunity by presenting on antigen-presenting cells such as dendritic cells. This ultimately links to the adaptive arm, where antibody production and maturation occur against that particular antigen. Upon binding with their specific antigens, antibodies trigger various immune responses to eliminate pathogens in a process called complement-dependent cytotoxicity and phagocytosis of invading microorganisms by immune cells or induce antibody-dependent cellular cytotoxicity is done by antibodies. These engineered antibodies are being used for various purposes, such as therapeutics, diagnostics, and biotechnology research. Cutting-edge techniques that include hybridoma technology, transgenic mice, display techniques like phage, yeast and ribosome displays, and next-generation sequencing are ways to engineer antibodies and mass production for the use of humankind. Considering the importance of antibodies in protecting from a diverse array of pathogens, investing in research holds great promise to develop future therapeutic targets to combat various diseases.

One-pot green synthesis of ZIF-8/IgG composite for the precise orientation and protection of antibody and its application in purification and detection of aflatoxins in peanut oil.

This study presents a novel approach toward the one-pot green synthesis of ZIF-8/IgG composite, focusing on its precise orientation and protection of the anti-aflatoxins antibody. The antibody orientation is achieved through the specific binding of IgG to the Fc region of the antibody, while the antibody protection is accomplished by the structural change restriction of ZIF-8 framework to the antibody. Consequently, the antibody exhibits enhanced target capability and significantly improved tolerance to organic solvents. The ZIF-8/IgG/anti-AFT was employed for the purification and detection of AFTs by coupling with UPLC. Under optimized conditions, the recoveries of spiked AFTs in peanut oils are between 86.1% and 106.4%, with relative standard deviations (RSDs) ranging from 0.8% to 8.8%. The linearity range is 0.5-20.0 ng for AFB1 and AFG1, 0.125-5.0 ng for AFB2 and AFG2, the limit of detection is 0.1 ng for AFB1 and AFG1, 0.03 ng for AFB2 and AFG2.

Natural antibodies targeting LPS in pleural effusions of various etiologies.

Respiratory infection, cancer, and heart failure can cause abnormal accumulation of fluid in the pleural cavity. The immune responses within the cavity are orchestrated by leucocytes that reside in the serosal-associated lymphoid tissue. Natural antibodies (NAbs) are abundant in the serum (S) having a major role in systemic and mucosal immunity; however, their occurrence in pleural fluid (PF) remains an open question. Our aim herein was to detect and measure the levels of NAbs (IgM, IgG, IgA) targeting lipopolysaccharides (LPS) in both the pleural fluid and the serum of 78 patients with pleural effusions (PEs) of various etiologies. The values of anti-LPS NAb activity were extracted through a normalization step regarding the total IgM, IgG, and IgA levels, all determined by in-house ELISA. In addition, the ratios of PF/S values were analyzed further with other critical biochemical parameters from pleural fluids. Anti-LPS NAbs of all Ig classes were detected in most of the samples, while a significant increase of anti-LPS activity was observed in infectious and noninfectious compared with malignant PEs. Multivariate linear regression confirmed a negative correlation of IgM and IgA anti-LPS PF/S ratio with malignancy. Moreover, anti-LPS NAbs PF/S measurements led to increased positive and negative predictive power in ROC curves generated for the discrimination between benign and malignant PEs. Our results highlight the role of anti-LPS NAbs in the pleural cavity and demonstrate the potential translational impact that should be further explored.NEW & NOTEWORTHY Here we describe the detection and quantification of natural antibodies (NAbs) in the human pleural cavity. We show for the first time that IgM, IgG, and IgA anti-LPS natural antibodies are detected and measured in pleural effusions of infectious, noninfectious, and malignant etiologies and provide clinical correlates to demonstrate the translational impact of our findings.

A general approach for selection of epitope-directed binders to proteins.

Directing antibodies to a particular epitope among many possible on a target protein is a significant challenge. Here, we present a simple and general method for epitope-directed selection (EDS) using a differential phage selection strategy. This involves engineering the protein of interest (POI) with the epitope of interest (EOI) mutated using a systematic bioinformatics algorithm to guide the local design of an EOI decoy variant. Using several alternating rounds of negative selection with the EOI decoy variant followed by positive selection on the wild-type POI, we were able to identify highly specific and potent antibodies to five different EOI antigens that bind and functionally block known sites of proteolysis. Among these, we developed highly specific antibodies that target the proteolytic site on the CUB domain containing protein 1 (CDCP1) to prevent its proteolysis allowing us to study the cellular maturation of this event that triggers malignancy. We generated antibodies that recognize the junction between the pro- and catalytic domains for three different matrix metalloproteases (MMPs), MMP1, MMP3, and MMP9, that selectively block activation of each of these enzymes and impair cell migration. We targeted a proteolytic epitope on the cell surface receptor, EPH Receptor A2 (EphA2), that is known to transform it from a tumor suppressor to an oncoprotein. We believe that the EDS method greatly facilitates the generation of antibodies to specific EOIs on a wide range of proteins and enzymes for broad therapeutic and diagnostic applications.

Facile generation of biepitopic antibodies with intrinsic agonism for activating tumor necrosis factor receptors.

Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.e., biparatopic) antibodies typically requires animal immunization and is laborious and unpredictable. Here, we report a simple method for identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses existing, receptor-specific IgGs, which lack intrinsic agonist activity, to block their corresponding epitopes, then selects single-chain antibodies that bind accessible epitopes. The selected antibodies are fused to the light chains of IgGs to generate human tetravalent antibodies. We highlight the broad utility of this approach by converting several clinical-stage antibodies against OX40 and CD137 (4-1BB) into biepitopic antibodies with potent agonist activity.

A new era of antibody discovery: an in-depth review of AI-driven approaches.

Given their high affinity and specificity for a range of macromolecules, antibodies are widely used in the treatment of autoimmune diseases, cancers, inflammatory diseases, and Alzheimer's disease (AD). Traditional experimental methods are time-consuming, expensive, and labor-intensive. Recent advances in artificial intelligence (AI) technologies provide complementary methods that can reduce the time and costs required for antibody design by minimizing failures and increasing the success rate of experimental tests. In this review, we scrutinize the plethora of AI-driven methodologies that have been deployed over the past 4 years for modeling antibody structures, predicting antibody-antigen interactions, optimizing antibody affinity, and generating novel antibody candidates. We also briefly address the challenges faced in integrating AI-based models with traditional antibody discovery pipelines and highlight the potential future directions in this burgeoning field.

Controlled silver electrodeposition on gold nanoparticle antibody tags for ultrasensitive prostate specific antigen sensing using electrochemical and optical smartphone detection.

One of the current challenges in medicine is to achieve a rapid and unequivocal detection and quantification of extremely low levels of disease biomarkers in complex biological samples. Here, we present the development and analytical evaluation of a low-cost smartphone-based system designed for ultrasensitive detection of the prostate-specific antigen (PSA) using two detection alternatives: electrochemical or optical, by coupling the smartphone with a portable potentiostat or magnifying lenses. An antibody tagged with gold nanoparticles (AuNPs), and indium tin oxide coated polyethylene terephthalate platform (ITO-PET) have been used to develop a sandwich-type immunoassay. Then, a controlled silver electrodeposition on the AuNPs surface is carried out, enhancing their size greatly. Due to such strong nanoparticle-size amplification (from nm to µm), the final detection can be dual, by measuring current intensity or the number of silver-enlarged microstructures generated. The proposed strategies exhibited limit detections (LOD) of 102 and 37 fg/mL for electrochemical and optical detection respectively. The developed immunosensor reaches excellent selectivity and performance characteristics to quantify biomarkers at clinically relevant values without any pretreatment. These proposed procedures could be useful to check and verify possible recurrence after clinical treatment of tumors or even report levels of disease serum biomarkers in early stages.

Formation and clinical effects of anti-drug antibodies against biologics in psoriasis treatment: An analysis of current evidence.

BACKGROUND: Formation of anti-drug antibodies (ADAs) against biologics is an important cause of psoriasis treatment failure. OBJECTIVE: This study aimed to summarize the characteristics of ADAs formation under different biological therapies and the influence of ADAs on the clinical effects and safety of biologics in patients with psoriasis. METHODS: PubMed, Embase, and Web of Science databases were searched from their inception to August 2022. Studies on biologics that assessed ADA levels in patients with psoriasis were included. The Cochrane risk-of-bias tool was used to assess the quality of randomized controlled trials (RCTs), the Newcastle-Ottawa Quality Assessment Scale (NOS) for case-control and cohort studies, and the Joanna Briggs Institute (JBI) critical appraisal checklist for single-arm studies. We calculated the pooled incidence with a random-effects model using R software. Subgroup analyses revealed that differences in patient characteristics, disease conditions, study design, and immunoassays may influence ADA generation and detection. RESULTS: The analysis included 86 studies, with a total population of 42,280 individuals. The pooled ADA rates were 0.49%, 2.20%, 2.38%, 4.08%, 7.38%, 7.94%, 14.29%, 21.93%, 29.70%, 31.76%, and 39.58% for secukinumab, etanercept, brodalumab, ustekinumab, tildrakizumab, guselkumab, ixekizumab, risankizumab, infliximab, adalimumab, and bimekizumab, respectively. >70% (95% CI, 0.71-0.81) of ADAs against adalimumab were neutralizing antibodies, and over 70% of ADAs against secukinumab and brodalumab were transient. Concomitant methotrexate therapy with tumor necrosis factor-α (TNF-α) inhibitors decreased ADA levels. Lower infliximab doses and intermittent therapy with interleukin (IL)-23 p19 inhibitors increased ADA formation. Additionally, ADA formation under treatment using TNF-α inhibitors and IL-12/23 p40 inhibitors was associated with lower response rates or serum drug levels, but only high ADA titers reduced the clinical effects of IL-17 inhibitors. The occurrence of IL-23 p19 and TNF-α inhibitors has been linked to injection-site reactions. CONCLUSIONS: Among the 11 biologics, secukinumab, etanercept, and brodalumab resulted in the lowest ADA formation rates. Immunogenicity contributes to lower biological efficacy and a higher likelihood of injection-site reactions. Low doses, intermittent treatment may increase ADA formation. An appropriate biologic should be selected based on the ADA formation rate and course of treatment.

Detection of estradiol with a digital immunoassay using an anti-immunocomplex antibody and single-molecule observation of gold nanoparticles.

Gold nanoparticles (AuNPs) have been widely applied to molecular sensors due to their optical properties. We previously reported a molecular detection by observing the scattered light of AuNPs at a single nanoparticle level using dark field microscopy (DFM). Recently, a molecular detection method using digital immunoassay has been reported, taking advantage of the characteristics of DFM. However, the digital immunoassays reported so far have been performed by a conventional sandwich immunoassay, which is difficult to apply to the detection of small molecules. In this study, with the aim of small molecule detection, we developed a digital immunoassay method using an anti-immunocomplex antibody that specifically recognizes immunocomplexes of small molecules with antibodies. The number of AuNPs modified with anti-immunocomplex antibody bound to immunocomplex of estradiol and anti-estradiol antibody was counted at a single nanoparticle level using DFM. We demonstrated for the first time that estradiol molecule can be detected by digital immunoassay using DFM and an anti-immunocomplex antibody with a detection sensitivity of 1 pg/mL.

Preclinical characterization of an mRNA-encoded anti-Claudin 18.2 antibody.

IMAB362/Zolbetuximab, a first-in-class IgG1 antibody directed against the cancer-associated gastric-lineage marker CLDN18.2, has recently been reported to have met its primary endpoint in two phase 3 trials as a first-line treatment in combination with standard of care chemotherapy in CLDN18.2-positive Her2 negative advanced gastric cancer. Here we characterize the preclinical pharmacology of BNT141, a nucleoside-modified RNA therapeutic encoding the sequence of IMAB362/Zolbetuximab, formulated in lipid nanoparticles (LNP) for liver uptake. We show that the mRNA-encoded antibody displays a stable pharmacokinetic profile in preclinical animal models, mediates CLDN18.2-restricted cytotoxicity comparable to IMAB362 recombinant protein and inhibits human tumor xenograft growth in immunocompromised mice. BNT141 administration did not perpetrate mortality, clinical signs of toxicity, or gastric pathology in animal studies. A phase 1/2 clinical trial with BNT141 mRNA-LNP has been initiated in advanced CLDN18.2-expressing solid cancers (NCT04683939).

Super-resolution imaging of antibody-conjugated biodegradable periodic mesoporous organosilica nanoparticles for targeted chemotherapy of prostate cancer.

Biodegradable periodic mesoporous organosilica nanoparticles (nanoPMOs) are widely used as responsive drug delivery platforms for targeted chemotherapy of cancer. However, the evaluation of their properties such as surface functionality and biodegradability is still challenging, which has a significant impact on the efficiency of chemotherapy. In this study, we have applied direct stochastic optical reconstruction microscopy (dSTORM), a single-molecule super-resolution microscopy technique, to quantify the degradation of nanoPMOs triggered by glutathione and the multivalency of antibody-conjugated nanoPMOs. Subsequently, the effect of these properties on cancer cell targeting, drug loading and release capability, and anticancer activity is also studied. Due to the higher spatial resolution at the nanoscale, dSTORM imaging is able to reveal the structural properties (i.e., size and shape) of fluorescent and biodegradable nanoPMOs. The quantification of nanoPMOs' biodegradation using dSTORM imaging demonstrates their excellent structure-dependent degradation behavior at a higher glutathione concentration. The surface functionality of anti-M6PR antibody-conjugated nanoPMOs as quantified by dSTORM imaging plays a key role in prostate cancer cell labeling: oriented antibody is more effective than random ones, while high multivalency is also effective. The higher biodegradability and cancer cell-targeting properties of nanorods conjugated with oriented antibody (EAB4H) effectively deliver the anticancer drug doxorubicin to cancer cells, exhibiting potent anticancer effects.

Chimeric HLA antibody receptor T cells to target HLA-specific B cells in solid organ transplantation.

HLA-sensitized patients on the transplant waiting list harbor antibodies and memory B cells directed against allogeneic HLA molecules, which decreases the chance to receive a compatible donor organ. Current desensitization strategies non-specifically target circulating antibodies and B cells, warranting the development of therapies that specifically affect HLA-directed humoral immune responses. We developed Chimeric HLA Antibody Receptor (CHAR) constructs comprising the extracellular part of HLA-A2 or HLA-A3 coupled to CD28-CD3ζ domains. CHAR-transduced cells expressing reporter constructs encoding T-cell activation markers, and CHAR-transduced CD8+ T cells from healthy donors were stimulated with HLA-specific monoclonal antibody-coated microbeads, and HLA-specific B cell hybridomas. CHAR T cell activation was measured by upregulation of T cell activation markers and IFNγ secretion, whereas CHAR T cell killing of B cell hybridomas was assessed in chromium release assays and by IgG ELISpot. HLA-A2- and HLA-A3-CHAR expressing cells were specifically activated by HLA-A2- and HLA-A3-specific monoclonal antibodies, either soluble or coated on microbeads, as shown by CHAR-induced transcription factors. HLA-A2 and HLA-A3 CHAR T cells efficiently produced IFNγ with exquisite specificity and were capable of specifically lysing hybridoma cells expressing HLA-A2- or HLA-A3-specific B-cell receptors, respectively. Finally, we mutated the α3 domain of the CHAR molecules to minimize any alloreactive T-cell reactivity against CHAR T cells, while retaining CHAR activity. These data show proof of principle for CHAR T cells to serve as precision immunotherapy to specifically desensitize (highly) sensitized solid organ transplant candidates and to treat antibody-mediated rejection after solid organ transplantation.

Individual recombinant repeats of MUC16 display variable binding to CA125 antibodies.

BACKGROUND: Despite its importance in the clinical management of ovarian cancer, the CA125 biomarker - located on the mucin protein MUC16 - is still not completely understood. Questions remain about MUC16's function and structure, specifically the identity and location of the CA125 epitopes. OBJECTIVE: The goal of this study was to characterize the interaction of individual recombinant repeats from the tandem repeat domain of MUC16 with antibodies used in the clinical CA125 II test. METHODS: Using E. coli expression, we isolated nine repeats from the putative antigenic domain of CA125. Amino acid composition of recombinant repeats was confirmed by high-resolution mass spectrometry. We characterized the binding of four antibodies - OC125, M11, "OC125-like," and "M11-like" - to nine recombinant repeats using Western blotting, indirect enzyme-linked immunosorbent assay (ELISA), and localized surface plasmon resonance (SPR) spectroscopy. RESULTS: Each recombinant repeat was recognized by a different combination of CA125 antibodies. OC125 and "OC125-like" antibodies did not bind the same set of recombinant repeats, nor did M11 and "M11-like" antibodies. CONCLUSIONS: Characterization of the interactions between MUC16 recombinant repeats and CA125 antibodies will contribute to ongoing efforts to identify the CA125 epitopes and improve our understanding of this important biomarker.