Search Results (232)

Background: Traumatic brain injury (TBI) affects millions of individuals annually and remains a major global cause of neurological disability and death. Tau protein hyperphosphorylation, particularly in its cis conformation, is a major pathological hallmark contributing to neurodegeneration following TBI. Single-chain variable fragments (scFvs), despite their diagnostic potential, suffer from rapid renal clearance and short circulation half-lives, which limit their in vivo performance. PEGylation is therefore employed to prolong systemic circulation and improve the pharmacokinetic behavior of scFvs, enabling more effective brain retention and target engagement. Methods: In this study, we utilized a previously validated anti-cis p-tau scFv antibody fragment, radiolabeled with technetium-99m tricarbonyl (^99mTc(CO)₃), as a diagnostic tracer to detect tau pathology in TBI rat models. The antibody was conjugated with polyethylene glycol (PEG, 20 kDa); PEGylation efficiency was determined by quantifying the products on SDS-PAGE, and the products were subsequently radiolabeled. Results: Radiochemical purity (RCP) was ~95.4% for the non-PEGylated tracer (^99mTc-AININ20) and ~92.7% for the PEGylated form (^99mTc-AININ20-PEG), with both showing >90% radiochemical purity consistently. Upon systemic administration, PEGylated scFv was able to cross the blood–brain barrier (BBB) and selectively accumulated in injured regions, as confirmed by single-photon emission computed tomography (SPECT) imaging. Both PEGylated and non-PEGylated scFv tracers showed significantly higher brain uptake in TBI rats compared to healthy controls (p < 0.0001). At 24 h, the PEGylated form exhibited a significantly higher brain signal than the non-PEGylated version (p < 0.0001), indicating improved tracer retention. Biodistribution analysis at 2 h post-injection showed significantly reduced renal clearance for the PEGylated tracer and increased hepatic uptake compared to the non-PEGylated form. At 24 h, in vivo imaging confirmed sustained brain retention, highlighting improved pharmacokinetics and imaging potential. Conclusions: These results support PEGylated scFv as a promising SPECT imaging agent for early detection of tauopathy in TBI, offering enhanced brain retention and improved pharmacokinetics. Full article

Saposin-like protein 2 (SAP2) exhibits strong immunogenicity as an antigen for immunodiagnosis in ruminant and human fasciolosis. Most available immunodiagnostic test kits are based on polyclonal and monoclonal antibodies against antigens from Fasciola spp. Previous studies demonstrated that polyclonal and monoclonal antibodies against SAP2 showed high specificity and could effectively detect Fasciola spp. infections at an early stage. However, polyclonal antibodies are extremely difficult to produce, and quality control is not possible during production; the procedure also involves considerable financial investment. To overcome these problems, we developed a single-chain variable fragment (scFv) to control quality in each production cycle and reduce the cost of manufacturing immunodiagnostic kits. Our objectives were to produce and characterize an scFv that binds the SAP2 from the liver fluke Fasciola gigantica. We constructed the scFv by genetic engineering: we cloned immunoglobulin genes and linked them with flexible polypeptide linkers composed of repeating glycine and serine residues. We selected an scFv with high affinity for binding SAP2 using the phage-display technique and produced it using a prokaryotic expression system. The scFv was characterized via in silico and in vitro methods to confirm its specificity for SAP2, including IMGT/V-QUEST, IMGT/Collier-de-Perles, HADDOCK 2.4, ELISA, immunoblotting, and immunohistochemistry. The scFv was successfully produced and purified using Ni-NTA affinity chromatography. The purified scFvFgSAP2 was approximately 27 kDa, as confirmed by SDS-PAGE and immunoblot analysis. An indirect ELISA and immunoblotting indicated that scFvFgSAP2 had strong reactivity with F. gigantica compared to other parasite species. Moreover, immunolocalization of scFvFgSAP2 confirmed that it binds specifically to natural SAP2 in the cecal epithelium cells of F. gigantica. Therefore, this scFv targeting SAP2 is an effective material and can be used to develop immunodiagnostic procedures. Full article

Mitochondria are essential organelles that perform irreplaceable functions in neurons. The degeneration of neurons in Alzheimer’s disease (AD) is associated with mitochondrial damage, and Tau pathology represents a significant pathogenic factor in AD. However, the relationship between Tau and mitochondrial dysfunction during neuronal degeneration remains unclear. In this study, we investigated the effects and mechanisms by which extracellular Tau aggregates induce neuronal mitochondrial damage and dysfunction. The results showed that extracellular Tau aggregates lead to structural damage of mitochondria in SH-SY5Y cells and disrupt mitochondrial homeostasis. Extracellular Tau aggregates can also cause mitochondrial oxidative stress and inhibit oxidative phosphorylation in SH-SY5Y cells. Concurrently, extracellular Tau aggregates promote neuronal death through an increase in cytochrome C, mtDNA leakage and activation of the cGAS/STING pathway. We also explored the effects of a single-chain variable fragment antibody (scFv T1) and found that scFv T1 alleviated mitochondrial damage and dysfunction by inhibiting the formation of Tau aggregates. These findings suggest that targeting Tau pathology may be crucial to address neuronal mitochondrial impairment and that reduction of the toxicity associated with extracellular Tau aggregates could help slow Tau pathology progression. Full article

Background: Transferrin receptor protein 1 (TfR1) plays a central role in cellular iron uptake and is frequently overexpressed in malignant tumor cells, rendering it an attractive target for tumor-directed therapy and drug delivery. Methods: A fully human single-chain variable fragment (scFv) antibody targeting TfR1, termed T8scFv, was isolated from a human scFv phage display library through three rounds of stringent biopanning and subsequently reformatted into a full-length IgG1 antibody (T8IgG1). Binding kinetics were characterized using Octet biolayer interferometry (BLI), while cellular binding and internalization were assessed by flow cytometry and immunofluorescence microscopy, respectively. T8IgG1 was further conjugated to DT3C, a recombinant truncated diphtheria toxin fusion protein, to evaluate its internalization-dependent cytotoxicity in vitro. Results: T8scFv exhibited nanomolar affinity for TfR1 (K_D = 214 ± 1 nM), which was substantially enhanced following conversion to the IgG1 format (T8IgG1, K_D = 18.5 ± 0.1 nM). T8IgG1 specifically recognized TfR1 on the surface of tumor cells and underwent efficient TfR1-mediated internalization. The T8IgG1-DT3C complex significantly reduced cell viability and induced apoptosis in K562 cells in vitro. Conclusions: These findings indicate that T8IgG1 is a moderate-affinity, internalizing anti-TfR1 antibody and highlight its potential as a promising candidate for TfR1-based targeted antitumor drug delivery systems. Full article

Malaria remains a major global health challenge. While treatments targeting parasite replication exist, effective interventions for neurological manifestations are scarce, necessitating new strategies for cerebral malaria. In this study, we investigated the effect of a single-chain variable fragment (scFv) against glycosylphosphatidylinositol (GPI) as an intervention tool to mitigate the effects of Plasmodium in a preclinical model. We used C57BL/6J mice infected with Plasmodium berghei-ANKA (PbA) and treated them with anti-GPI scFv or phosphate-buffered saline (PBS) on days 0, 3, and 6 post-infection. Uninfected controls were treated on the same days with scFv or PBS. The animals were evaluated for morbidity and mortality, body weight, parasitemia, blood count, cytokines, and histopathology. Results show that anti-GPI scFv prevented lethality in 71.4% of infected animals and promoted recovery from weight loss. Furthermore, the intervention inhibited neurological and systemic signs, reduced parasitemia, and improved hematological and histopathological parameters in the brain, lungs, and kidneys. In conclusion, anti-GPI scFv exerts a significant systemic effect on experimental cerebral malaria (ECM) pathology, representing a promising tool for severe manifestations of the disease. Full article

Despite the clinical success of antibody–drug conjugates (ADCs), their efficacy in solid tumors remains constrained by limited tumor penetration of the IgG format. Smaller antibody fragment–drug conjugates (FDCs) present a compelling alternative, potentially offering superior intratumoral distribution and a wider therapeutic window driven by rapid systemic clearance. This study compares therapeutic activity of ganglioside GD2-specific minibody–drug conjugates against full-length ch14.18 antibody–drug conjugates, and biodistribution of the respective minibody (scFv-C_H3 homodimer) and IgG formats in the GD2-positive B78-D14 melanoma syngeneic mouse model. We conjugated the minibody and antibody with MMAE or MMAF via a cathepsin-cleavable linker, generating FDCs with drug–antibody ratio (DAR) of 2 and ADCs with DAR of 2 or 4. The biodistribution analysis showed no significant difference in tumor uptake for both formats early in the analysis (2–4 h) and a higher tumor uptake for the IgG at 24 h post-injection. However, the minibody achieved a superior tumor-to-blood ratio (TBR) at all timepoints, reaching a TBR > 1 compared to ~0.2 for the antibody by 24 h. In vitro studies demonstrated higher cytotoxicity for the ADCs regardless of drug load (DAR 2 or 4) compared to the FDCs, although the difference between conjugates with equal DAR was modest in B78-D14 cells. Critically, superior in vitro ADC potency did not translate in vivo. Minibody–MMAF and minibody–MMAE achieved 74% and 55% tumor growth inhibition, respectively, by the study endpoint—demonstrating comparable efficacy to ADCs with twice the drug load when administered to mice at equimass dosing. Stron/g in vivo efficacy of anti-GD2 FDCs, combined with the superior TBR for the minibody format, underscores the potential of minibody–drug conjugates for treating GD2-positive tumors, particularly when ADC-associated toxicity precludes high-dose regimens. Full article

Canine distemper virus (CDV) remains a highly contagious and lethal pathogen, posing a severe global threat to domestic dogs and wild carnivores. To address the urgent need for effective interventions, we utilized a proprietary Vero-SLAM cell platform to isolate a wild-type CDV strain and generate neutralizing polyclonal antibodies. Subsequently, phage display technology was employed to screen for single-chain variable fragments (scFvs) targeting the CDV hemagglutinin protein (CDV-H). This approach led to the identification of a specific scFv with virus-binding affinity comparable to commercial antibodies, which effectively blocks CDV infection in Vero-SLAM cells. Molecular docking and molecular dynamics simulations were conducted to elucidate the interaction mechanism, suggesting that this scFv binds to a novel and unique epitope on the CDV-H. These findings not only expand our understanding of the antigenic properties of the CDV H protein but also provide a theoretical foundation and a promising candidate molecule for the development of future CDV diagnostics and antiviral strategies. Full article

Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) co-circulate widely and cause significant morbidity, yet effective broad-spectrum antivirals are limited. This study evaluated the antiviral efficacy, cytotoxicity, and host transcriptional responses to the nucleic acid–hydrolyzing antibody fragment 3D8 scFv in mono- and co-infection models. RNA sequencing of A549 cells treated with 3D8 scFv revealed a dose-dependent activation of the MAPK–HSP70 stress response, with minimal transcriptomic disruption at antiviral concentrations. Comparative transcriptomic analysis identified distinct host signatures for ZIKV and DENV2, and machine learning classifiers accurately distinguished infection states (AUC > 0.95). In Vero E6 cells, prophylactic treatment with 3D8 scFv significantly reduced viral RNA, protein expression, and infectious particle production for both viruses, including during co-infection. Optimized post-entry treatment also demonstrated antiviral activity. Cytotoxicity assays confirmed good tolerability at effective concentrations. These findings indicate that 3D8 scFv inhibits viral replication through early cleavage of viral nucleic acids while inducing a limited protective stress response, supporting its development as a broad-spectrum antiviral candidate. Full article

Immunotoxins are chimeric molecules with high potential as therapeutic candidates that combine antibody specificity to recognize and bind tumor-associated antigens and the cytotoxic potency of the enzymatic activity of a toxin, leading to the selective death of target cells. The use of immunotoxins as therapeutic tools remains limited by various issues, such as selecting the appropriate tumor-associated antigen (TAA), penetration difficulties in solid tumors, low renal clearance, and low toxic payload. For this purpose, in this work we have designed a novel trimeric immunotoxin (IMTXTriA33αS) against colorectal cancer, combining the scFv against GPA33 as a targeting domain and the fungal ribotoxin α-sarcin (αS) as the toxic fragment, linked by a trimerization domain (TIE^XVIII). Our results demonstrate that IMTXTriA33αS has greater avidity and toxic load, showing a very significant increase in its in vitro and in vivo antitumor efficacy, due to its trimeric structure. Full article

Background: Melanoma and triple-negative breast cancer (TNBC) are the most aggressive skin and breast cancers, often diagnosed at late stages with limited treatment options. The melanoma-associated antigen melanotransferrin (MTf) is overexpressed in these solid tumors, where it drives tumorigenesis, progression, and chemoresistance. Its inhibition correlates with tumor regression, making MTf a promising therapeutic target. This study aimed to develop a novel, selectively targeted antibody–drug conjugate (ADC) against MTf-expressing melanoma and TNBC cancer cells using SNAP-tag fusion protein conjugation technology. Methods: We generated an L49(scFv)-SNAP-tag antibody fusion protein engineered through the genetic fusion of a humanized anti-MTf single-chain variable fragment (scFv) with a SNAP-tag fusion protein capable of site-specific self-labelling with O⁶-benzylguanine (BG) modified substrates in 1:1 stoichiometry. Binding and internalization of the conjugate labeled with BG-Alexa 488 (L49(scFv)-SNAP-Alexa488) were assessed by confocal microscopy and flow cytometry in MTf-overexpressing cell lines. Cytotoxicity was evaluated using the cell viability XTT assay after conjugating the SNAP-fusion protein to the potent monomethyl auristatin-F (BG-AURIF). Results: The L49(scFv)-SNAP-Alexa488 conjugate demonstrated specific binding and internalization into MTf-positive melanoma and TNBC cells. The corresponding ADC, L49(scFv)-SNAP-Linker-AURIF, exerted potent, antigen and dose-dependent cytotoxicity, with IC₅₀ values in the nanomolar range (4.77–34.43 nM). Conclusions: We successfully generated a novel SNAP-tag-based ADC that selectively eliminates MTf-overexpressing tumor cells. This proof-of-concept highlights MTF’s value as a therapeutic target and demonstrates that a smaller-format, non-cleavable linker SNAP-tag-based ADC can achieve potent nanomolar cytotoxicity, supporting further development of MTF-targeted immunotherapies for melanoma and TNBC. Full article

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

Adoptive cell therapy (ACT) with T cells modified with a chimeric antigen receptor (CAR T cells) has dramatically improved outcomes in hematologic cancers. However, its efficacy in solid tumors, such as melanoma, is hampered by several factors. These include heterogeneous expression of tumor-associated antigens (TAA) and an immunosuppressive, profibrotic tumor microenvironment (TME), which restricts cytotoxic CAR T cells trafficking into the tumor, as well as their persistence and cytolytic activity. As a result, responses to CAR T cell monotherapy in melanoma and other solid tumors are typically weak, transient or even absent. Emerging evidence suggests that combining traditional chemotherapy with CAR T cell therapy can enhance the antitumor activity of CAR T cells in solid malignancies. Partial tumor cell killing by chemotherapy improves access to TAA and disrupts the TME by affecting the global structure of the tumor tissue. Here, we developed an immunocompetent syngeneic B16 melanoma mouse model to test a combination of classical dacarbazine (DTIC) chemotherapy with ACT with murine CAR T cells. B16-F10 (next as B16) melanoma cells were modified to express a human/murine hybrid epidermal growth factor receptor (EGFR) recognized by a murine CAR bearing a single-chain variable fragment (scFv) derived from cetuximab, an anti-EGFR monoclonal antibody approved for the treatment of colorectal and certain other solid tumors. Prior to CAR T cells administration, cyclophosphamide (CPA) pre-conditioning was used. We demonstrated that DTIC therapy followed by infusion of murine CAR T cells targeting the human/murine hybrid EGFR (EGFR mCAR T cells) provided superior tumor control and prolonged survival compared to monotherapy with either DTIC or EGFR mCAR T cells alone. These findings support the potential feasibility of a combined therapeutic strategy for human melanoma involving DTIC treatment followed by EGFR CAR T cells infusion after CPA pre-conditioning. 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

Multiple myeloma (MM) is an incurable hematologic malignancy arising from clonal plasma cells, with poor long-term outcomes due to inevitable relapse after conventional therapies. Chimeric antigen receptor (CAR) T-cell immunotherapy targeting B-cell maturation antigen (BCMA) has shown remarkable efficacy in relapsed patients. Conventional CARs employ single-chain variable fragments (scFvs), whereas single-domain antibodies (sdAb or VHHs) offer advantages such as small size, high stability, and potentially reduced immunogenicity. We designed and evaluated a novel anti-BCMA nanoCAR-T based on the VHH Nb17, compared with the conventional scFv-based CAR-T CT103a. Nb17 demonstrated strong BCMA binding and was incorporated into a CAR construct. Both nanoCAR-T and CT103a were generated via lentiviral transduction of primary T cells. Their cytotoxicity, cytokine secretion, degranulation, memory phenotype, and gene expression were assessed in vitro, along with antitumor activity in vivo. Nb17-nanoCAR-T demonstrated specific cytotoxicity, cytokine release (IL-2, TFNa, IFNg), and CD107a degranulation comparable to CT103a. Transcriptomic analysis revealed overlapping pathways between both CARs. Upon rechallenge, both CARs showed enhanced proliferation compared with untransduced T cells. In vivo, Nb17-nanoCAR-T and CT103a eradicated tumors in NSG mice. These findings demonstrate Nb17-nanoCAR-T exhibits potent anti-myeloma efficacy comparable to scFv-based CAR-T, supporting its potential as a promising therapeutic alternative. Full article

Chimeric antigen receptor (CAR) T cell therapy for B cell malignancies is often limited by T cell exhaustion, which is frequently driven by the PD-1/PD-L1 immune checkpoint axis. To overcome this, we developed an “armored” CAR-T cell strategy using a novel bidirectional promoter system. We engineered a single vector to co-express a CD19-specific CAR alongside a secreted anti-PD1 molecule, in either a full-length antibody or a single-chain variable fragment (scFv) format, using the Sleeping Beauty (SB) transposon system. The sequences for the anti-PD1 modules were derived from the clinical antibody nivolumab. Both armored constructs demonstrated robust CAR expression, comparable to or higher than conventional CAR-T cells, and proliferated significantly more than untransfected controls. The engineered cells successfully secreted their anti-PD1 payloads, with the full-length antibody showing more sustained secretion than the scFv. This autocrine blockade resulted in significantly reduced surface PD1 expression on the armored CAR-T cells. Functionally, the anti-PD1-secreting cells exhibited superior cytotoxicity against PD-L1-positive Raji target cells, particularly at low effector-to-target ratios. Critically, in a serial rechallenge assay designed to simulate chronic antigen exposure, both armored CAR-T cell groups showed markedly enhanced proliferation and persistence compared to conventional CAR-T cells, which failed to expand after repeated stimulation. Our findings validate the bidirectional EF1 promoter as an efficient system for generating multi-functional T cells and demonstrate that armoring CAR-T cells with secreted anti-PD1 antibodies is a potent strategy to enhance their persistence and anti-tumor efficacy. Full article