Search Results (29)

Metalloproteinases (MPs) are zinc-dependent endopeptidases, including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs), implicated in various diseases such as cancer, neurodegenerative disorders, and cardiovascular conditions. Among MPs, ADAM-17, also known as tumor necrosis factor-α (TNF-α)-converting enzyme (TACE), plays a crucial role in extracellular matrix remodeling and cytokine release. Dysregulation of ADAM-17 contributes to inflammatory diseases, cancer progression, and immune modulation. While small-molecule inhibitors have been limited by off-target effects and instability, antibody-based approaches offer a more selective strategy. Monoclonal antibodies show promise in blocking ADAM-17 activity, but there are concerns about toxicity due to the lack of selectivity. Enhancing the binding affinity and selectivity of single-chain antibodies requires unraveling the structural details that drive MP targeting. This study uses yeast surface display (YSD) and fluorescence-activated cell sorting (FACS) to engineer single-chain variable fragment (scFv) antibodies with optimized complementarity-determining region 3 of the heavy chain (CDR-H3) conformations. Next-generation sequencing (NGS) was used to identify key residues contributing to high-affinity ADAM-17 binding. These findings offer a framework for designing monoclonal antibodies against ADAM-17 and other MPs, paving the way for novel antibody-based designer scaffolds with applications in developing therapeutics. Full article

Cervical cancer (CC) can be prevented through continuous screening and the timely detection of cervical intraepithelial neoplasia (CIN) using immunohistochemistry techniques to identify biomarker expressions. In a previous study, we proposed nuclear REST loss as a biomarker in precancerous lesions and CC; however, no validated antibodies are available for detecting REST in cytology or cervical tissues. Although we have developed an IgM-type anti-REST monoclonal antibody capable of detecting REST in liquid-based cytology cells, it was not useful for the detection of REST in cervical tissues by immunohistochemistry. The main objective of this study is to generate single-chain variable fragments (scFvs) for the clinical evaluation of REST in cervical tissues from women with CIN and CC. Using RNA from an IgM-producing hybridoma anti-REST, we conducted RT-PCR and PCR to obtain the coding sequences for the variable regions of the heavy and light chains. These sequences were joined with a linker to create a single-chain antibody. The scFv was then cloned into the pSyn1 vector, expressed in E. coli TG1, and purified through chromatography. Subsequently, it was characterized using immunological methods to assess its biological activity and employed to evaluate REST expression in cytological samples and cervical tissues. The anti-REST scFv represents an innovative detection tool that retains the antigen recognition of the parental IgM while overcoming its size limitation, enabling tissue penetration and detection of REST in cervical samples. Its application facilitates the identification of REST in cervical samples, reinforcing REST’s potential as a diagnostic biomarker for CC and CIN. Full article

Sepsis is a fetal disease that requires a clear diagnostic biomarker for timely antibiotic treatment. Recent research has identified a pyroptosis-inducing epitope known as P5-5 in tetranectin (TN), a plasma protein produced by monocytes. Previously, we produced a 12F1 monoclonal antibody against the P5-5 and discovered that it could not only diagnose the presence but also monitor the progress of sepsis in the clinic. In the current study, we further investigated the structure site of the P5-5 and the recognition mechanism between the 12F1 mAb and the P5-5 epitope. To this end, 10 amino acids (NDALYEYLRQ) in the P5-5 were individually mutated to alanine, and their binding to the mAb was tested to confirm the most significant antigenic recognition sites. In the meanwhile, the spatial conformation of 12F1 mAb variable regions was modeled, and the molecular recognition mechanisms in detail of the mAb to the P5-5 epitope were further studied by molecular docking. Following epitope prediction and experimental verification, we demonstrated that the motif “DALYEYL” in the epitope sequence position 2−8 of TN-P5-5 is the major binding region for mAb recognition, in which two residues (4L and 8L) were essential for the interaction between the P5-5 epitope and the 12F1 mAb. Therefore, our study greatly narrowed down the previously reported motif from ten to seven amino acids and identified two Leu as critical contact residues. Finally, a single-chain variable fragment (scFv) from the 12F1 hybridoma was constructed, and it was confirmed that the identified motif and residues are prerequisites for the strong binding between P5-5 and 12F1. Altogether, the data of the present work could serve as a theoretic guide for the clinical design of biosynthetic drugs by artificial intelligence to treat sepsis. Full article

Background: Monoclonal antibodies (mAbs) are potent treatment options for infectious diseases. The rapid isolation and in vivo validation of therapeutic mAb candidates, including mAb cocktails, are essential to combat novel or rapidly mutating pathogens. The rapid selection and production of mAb candidates in sufficient amount and quality for preclinical studies are a major limiting step in the mAb development pipeline. Methods: Here, we developed a method to facilitate the screening of therapeutic mAbs in mouse models. Four conventional mAbs were transformed into single-chain variable fragments fused to the fragment crystallizable (Fc) region of a human IgG1 (scFv-IgG). These scFv-IgG were expressed individually or as a cocktail in vitro and in mice following transfection or hydrodynamic delivery of the corresponding plasmids. Results: This method induced high expression of all scFv-IgG and provided protection in two murine infection models. Conclusions: This study highlights the benefits of this approach for the rapid, low-cost screening of therapeutic mAb candidates. Full article

Snakebite is a critical global public health issue, causing substantial mortality and morbidity, particularly in tropical and subtropical regions. The development of innovative antivenoms targeting snake venom toxins is therefore of paramount importance. In this study, we adopted an epitope-directed approach to design three degenerate 15-mer peptides based on amino acid sequence alignments of snake venom phospholipase A₂s (PLA₂s) and snake venom serine proteases (SVSPs) from snake (Crotalus atrox). By leveraging their immunogenic and inhibitory profiles, these peptides were specifically designed to target the Asp49 and Lys49 variants of PLA₂ and SVSP toxins. Groups of five mice were immunized with each peptide, and IgG mRNA was subsequently extracted from peripheral blood mononuclear cells (PBMCs) and spleen lymphocytes of the top three responders. The extracted mRNA was reverse-transcribed into complementary DNA (cDNA), and the variable regions of the IgG heavy and kappa chains were amplified using polymerase chain reaction (PCR). These amplified regions were then linked with a 66-nucleotide spacer to construct single-chain variable fragments (scFvs). Sequence analysis of 48 randomly selected plasmids from each PLA₂ and SVSP scFv library revealed that over 80% contained scFv sequences with notable diversity observed in the complementarity-determining regions (CDRs), particularly CDR3. Enzyme-linked immunosorbent assay (ELISA) results demonstrated that the SP peptide elicited a broader immune response in mice compared to the Asp49 peptide, implying the strong immunogenicity of the SP peptide. These scFvs represent a promising foundation for the development of recombinant human monoclonal antibodies targeting snake PLA₂ and SVSP toxins, providing a potential therapeutic strategy for the treatment of snakebites. Full article

Osteocalcin is a useful biomarker for bone formation and bone-related diseases. KTM219 is an anti-osteocalcin C-terminal peptide antibody. The single-chain variable region (scFv) and antigen-binding fragment (Fab) of KTM219 are applicable to the Quenchbody (Q-body) immunoassay. Q-body is a new type of fluorescent immunosensor, which is scFv or Fab labeled with a fluorescent dye. When Q-body binds to its antigen, the fluorescence intensity increases. The highly sensitive detection of antigens by changes in fluorescence intensity is performed in a single step by mixing the sample and reagent. In this study, to reveal the recognition mechanism of the KTM219 antibody and to discuss the structural basis for Q-body, we solved the crystal structures of Fab of the anti-osteocalcin antibody KTM219 and its complex with the antigen osteocalcin C-terminal peptide (BGP-C7). Also, we solved the structure of a KTM219 Fab crystal grown in the presence of a fluorescent dye, carboxytetramethylrhodamine (TAMRA); however, tightly bound TAMRA was not found in the electron density map. We predicted the binding sites of TAMRA in the antigen-binding pocket by docking simulations. These results support the proposed Q-body mechanism. The crystal structures of KTM219 Fab would be useful for further development and improvement of Q-body fluorescent immunosensors. Full article

Hen’s egg allergy is the second most common food allergy among infants and young children. The possible presence of undeclared eggs in foods poses a significant risk to sensitized individuals. Therefore, reliable egg allergen detection methods are needed to ensure compliance with food labeling and improve consumer protection. This work describes for the first time the application of phage display technology for the generation of a recombinant antibody aimed at the specific detection of hen’s ovomucoid. First, a single-chain variable fragment (scFv) library was constructed from mRNA isolated from the spleen of a rabbit immunized with ovomucoid. After rounds of biopanning, four binding clones were isolated and characterized. Based on the best ovomucoid-binding candidate SR-G1, an indirect phage enzyme-linked immunosorbent assay (phage-ELISA) was developed, reaching limits of detection and quantitation of 43 and 79 ng/mL of ovomucoid, respectively. The developed ELISA was applied to the analysis of a wide variety of food products, obtaining a good correlation with a commercial egg detection assay used as a reference. Finally, in silico modeling of the antigen-antibody complex revealed that the main interactions most likely occur between the scFv heavy chain and the ovomucoid domain-III, the most immunogenic region of this allergen. Full article

Enterovirus A71 (EV-A71) is one of the causative agents of hand-foot-mouth disease, which can be associated with neurocomplications of the central nervous system. A limited understanding of the virus’s biology and pathogenesis has led to the unavailability of effective anti-viral treatments. The EV-A71 RNA genome carries type I internal ribosomal entry site (IRES) at 5′ UTR that plays an essential role in the viral genomic translation. However, the detailed mechanism of IRES-mediated translation has not been elucidated. In this study, sequence analysis revealed that the domains IV, V, and VI of EV-A71 IRES contained the structurally conserved regions. The selected region was transcribed in vitro and labeled with biotin to use as an antigen for selecting the single-chain variable fragment (scFv) antibody from the naïve phage display library. The so-obtained scFv, namely, scFv #16-3, binds specifically to EV-A71 IRES. The molecular docking showed that the interaction between scFv #16-3 and EV-A71 IRES was mediated by the preferences of amino acid residues, including serine, tyrosine, glycine, lysine, and arginine on the antigen-binding sites contacted the nucleotides on the IRES domains IV and V. The so-produced scFv has the potential to develop as a structural biology tool to study the biology of the EV-A71 RNA genome. Full article

Recent advances in the use of gold nanoparticles (Au NPs)/antibody conjugations in nanomedicine have increased the need to optimize the synthesis conditions and surface functionalization of Au NPs. In this study, a home-made Neisseria meningitidis recombinant antibody (scFv-Fc) was developed by connecting the fragment crystallizable (Fc) region of a human antibody with a mouse recombinant antibody (single-chain variable fragment antibody (scFv)) and characterized using the SOEing PCR technique. Then, an optimized gold coating agent for the scFv-Fc/Au NP conjugation (i.e., the citrate agent) was found among three common agents (citrate, allylamine hydrochloride, and polyvinyl alcohol) with different surface charges (negative, positive, and neutral, respectively). Moreover, the stability of the scFv-Fc/protein A-G in the presence of a N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) linker was investigated using the docking method. It was found that the designed scFv-Fc/protein A-G/SPDP/citrate recombinant antibody showed optimized bottom-on conjugation of the protein A-G with the improved scFv-Fc/Au NPs, enabling a suitable interaction with the Neisseria meningitidis bacterial antigen. Full article

Our laboratory has identified and developed a unique human-engineered domain (HED) structure that was obtained from the human Alpha-2-macroglobulin receptor-associated protein based on the three-dimensional structure of the Z-domain derived from Staphylococcal protein A. This HED retains µM binding activity to the human IgG1CH2-CH3 elbow region. We determined the crystal structure of HED in association with IgG1’s Fc. This demonstrated that HED preserves the same three-bundle helix structure and Fc-interacting residues as the Z domain. HED was fused to the single chain variable fragment (scFv) of mAb 4D5 to produce an antibody-like protein capable of interacting with the p185Her2/neu ectodomain and the Fc of IgG. When further fused with murine IFN-γ (mIFN-γ) at the carboxy terminus, the novel species exhibited antitumor efficacy in vivo in a mouse model of human breast cancer. The HED is a novel platform for the therapeutic utilization of engineered proteins to alleviate human disease. Full article

Despite therapeutic advances in recent years, there are still unmet medical needs for patients with multiple myeloma (MM). Hence, new therapeutic strategies are needed. Using phage display for screening a large repertoire of single chain variable fragments (scFvs), we isolated several candidates that recognize a heavily sulfated MM-specific glycoform of the surface antigen syndecan-1 (CD138). One of the engineered scFv-Fc antibodies, named MM1, activated NK cells and induced antibody-dependent cellular cytotoxicity against MM cells. Analysis of the binding specificity by competitive binding assays with various glycan ligands identified N-sulfation of glucosamine units as essential for binding. Additionally, site-directed mutagenesis revealed that the amino acids arginine and histidine in the complementarily determining regions (CDRs) 2 and 3 of the heavy chain are important for binding. Based on this observation, a heavy-chain antibody, known as a nanobody, and a peptide mimicking the CDR loop sequences were designed. Both variants exhibited high affinity and specificity to MM cells as compared to blood lymphocytes. Specific killing of MM cells was achieved by conjugating the CDR2/3 mimic peptide to a pro-apoptotic peptide (KLAKLAK)_2. In a co-culture model, the fusion peptide killed MM cells, while leaving normal peripheral blood mononuclear cells unaffected. Collectively, the development of antibodies and peptides that detect tumor-specific glycoforms of therapeutic targets holds promise for improving targeted therapies and tumor imaging. Full article

Koi herpesvirus (KHV) is a highly contagious virus that causes high mortality in koi and common carp, leading to a reduction in production worldwide. Recent diagnostic tests based on molecular methods alone (nucleic acid amplification) and indirect immunoassay methods (antibody detection) can be confirmed over KHV infections or prior exposure and latent infections. Unfortunately, there is no established method to detect KHV virus particles, especially when virus titers are low. Therefore, we propose an alternative, direct immunoassay method for viral detection using a single-chain variable fragment (scFv), a specific region of IgG antibodies that binds specifically to KHV particles. The results of functional analyses indicated that four putative scFv candidates, C5, F8, F6, and E4, were specific to KHV, but only F6 and C5 had a high binding affinity. The binding characteristics were confirmed by indirect competitive and sandwich enzyme-linked immunosorbent assays, which indicated that F6 and C5 have a broad penetration area to the binding region and share a similar epitope with commercial KHV monoclonal antibodies. These characteristics were further confirmed by their interactions with purified KHV coat protein by indirect ELISA and Western blot analyses. In conclusion, the F6 and C5 scFvs have adequate binding affinity to KHV particles to permit their use in immunoassays. Full article

Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 10⁶ and 1.3 × 10⁷ transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously. Full article

Single-chain variable fragments (scFvs) have been recognized as promising agents in cancer therapy. However, short serum half-life of scFvs often limits clinical application. Fusion to albumin affibody (ABD) is an effective and convenient half-life extension strategy. Although one terminus of scFv is available for fusion of ABD, it is also frequently used for fusion of useful moieties such as small functional proteins, cytokines, or antibodies. Herein, we investigated the internal linker region for ABD fusion instead of terminal region, which was rarely explored before. We constructed two internally ABD-inserted anti-HER2 4D5scFv (4D5-ABD) variants, which have short (4D5-S-ABD) and long (4D5-L-ABD) linker length respectively. The model structures of these 4D5scFv and 4D5-ABD variants predicted using the deep learning-based protein structure prediction program (AlphaFold2) revealed high similarity to either the original 4D5scFv or the ABD structure, implying that the functionality would be retained. Designed 4D5-ABD variants were expressed in the bacterial expression system and characterized. Both 4D5-ABD variants showed anti-HER2 binding affinity comparable with 4D5scFv. Binding affinity of both 4D5-ABD variants against albumin was also comparable. In a pharmacokinetic study in mice, the 4D5-ABD variants showed a significantly prolonged half-life of 34 h, 114 times longer than that of 4D5scFv. In conclusion, we have developed a versatile scFv platform with enhanced pharmacokinetic profiles with an aid of deep learning-based structure prediction. Full article

Antibody discovery by phage display consists of two phases, i.e., the binding phase and the amplification phase. Ideally, the selection process is dominated by the former, and all the retrieved clones are amplified equally during the latter. In reality, the amplification efficiency of antibody fragments varies widely among different sequences and, after a few rounds of phage display panning, the output repertoire often includes rapidly amplified sequences with low or no binding activity, significantly diminishing the efficiency of antibody isolation. In this work, a novel synthetic single-chain variable fragment (scFv) library with complementarity-determining region (CDR) diversities aimed at improved amplification efficiency was designed and constructed. A previously reported synthetic scFv library with low, non-combinatorial CDR diversities was panned against protein A superantigen, and the library repertoires before and after the panning were analyzed by next generation sequencing. The enrichment or depletion patterns of CDR sequences after panning served as the basis for the design of the new library. Especially for CDR-H3 with a higher and more random diversity, a machine learning method was applied to predict potential fast-amplified sequences among a simulated sequence repertoire. In a direct comparison with the previous generation library, the new library performed better against a panel of antigens in terms of the number of binders isolated, the number of unique sequences, and/or the speed of binder enrichment. Our results suggest that the amplification-centric design of sequence diversity is a valid strategy for the construction of highly functional phage display antibody libraries. Full article