Search Results (921)

Background: Antibody–drug conjugates (ADCs) have evolved from early heterogeneous constructs into a mature therapeutic platform with exponential clinical relevance. This review highlights recent advances in ADC design and development, with emphasis on antigen selection, antibody engineering, linker and payload innovation, site-specific conjugation, clinical translation, toxicity, resistance, and emerging data-driven approaches. Methods: The review draws on the literature published from 2019 to the recent clinical and regulatory developments relevant to approved and late-stage ADCs, emphasizing the advances in target biology, antibody formats, linker chemistry, payload classes, conjugation technologies, developability assessment, and computational or artificial intelligence-assisted design strategies. Results: ADC development has evolved with improved target selection, enhanced internalization and tumor selectivity, and the use of engineered, bispecific, biparatopic, and fragment-based antibody formats. Linker and payload innovation has expanded beyond traditional microtubule inhibitors to include topoisomerase I inhibitors, DNA-damaging agents, and emerging dual-payload or non-cytotoxic strategies. Site-specific conjugation and improved control of drug-to-antibody ratio have increased stability, pharmacokinetic performance, and manufacturability. Clinically, ADCs are being used across a broader range of malignancies and treatment settings, although toxicities and resistance mechanisms remain an important limitations. Computational methods and artificial intelligence are increasingly being explored for target discovery, molecular optimization, toxicity prediction, and model-informed clinical development. Conclusions: ADCs are transitioning toward a more integrated, design-driven platform in which antigen biology, antibody format, chemistry, and computational prediction are jointly optimized. Future progress will depend on improved standardization, biomarker-guided development, and interdisciplinary approaches to enhance its therapeutic index and expand its applications beyond oncology. Full article

Background/Objectives: The use of cross-linking enzymes for site-selective and efficient antibody modification has attracted considerable attention. Microbial transglutaminase (MTG)-mediated labeling of IgG at Gln295 has emerged as a promising strategy for preparing antibody–drug conjugates (ADCs). By contrast, selective modification of a specific Lys residue on native antibody surfaces using MTG remains challenging because most Lys residues exhibit low intrinsic reactivity. Here, we address this challenge by exploiting enzyme–antibody proximity together with screening for highly reactive Gln-donor substrates from a random peptide library. Methods: Reactive Gln-donor peptide substrates were first identified from a seven-amino-acid phage-displayed peptide library using a reactive Lys-containing peptide as bait. Based on the obtained sequence, an azide-functionalized Gln-donor peptide suitable for click chemistry was designed. Results: The designed substrate enabled efficient Lys65-selective modification of Fab fragments using a fusion of an engineered MTG zymogen and protein G (EzMTG-pG), followed by functionalization through click chemistry to yield fluorescent Fab conjugates. Conclusions: These results provide practical guidelines for substrate design in MTG-mediated site-selective protein modification. Full article

African swine fever in both domestic and wild pig populations is caused by the extremely infectious African swine fever virus (ASFV). It seriously endangers biodiversity and results in large financial losses for the worldwide pork sector. The major capsid protein p72 is molecularly chaperoned by the ASFV pB602L protein, which is essential to viral assembly. Furthermore, as a nonstructural protein expressed at late stages of infection, pB602L induces a distinct antibody response that may complement existing serological assays based on structural proteins. Given its strong immunogenicity, pB602L represents a promising antigen for developing supplementary diagnostic tools for African swine fever (ASF). In this study, we successfully generated and separated the ASFV pB602L protein, and we verified its responsiveness using serum from pigs infected with ASFV. Additionally, we produced four monoclonal antibody-specific hybridoma cell lines that targeted the pB602L protein exclusively. These cell lines demonstrated high immunoreactivity and responsiveness toward ASFV pB602L. These results highlight the potential enhancement of diagnostic skills. We have detected two previously unknown linear B-cell epitopes (¹³⁸TIDSFL¹⁴³ and ¹⁶⁴TNVDTC¹⁶⁹) using overlapping peptide and truncated protein fragment analysis. Due to their high degree of conservation across various ASFV strains, these epitopes offer trustworthy candidates for the creation of particular diagnostic instruments. This study expands the known ASFV antigenic repertoire by systematically mapping immunodominant epitopes of pB602L. The identified epitopes provide potential molecular targets for the rational design of multi-epitope subunit vaccines. Full article

Immunoglobulins exhibit important biological functions, including the neutralization of cytotoxins, enhancement of phagocytic activity, and activation of the complement system, which have driven their widespread application in both the food and pharmaceutical industries. Due to their low cost and short production cycles, microbial expression systems such as bacteria and yeast have been increasingly developed in recent years for immunoglobulin production. However, microbial systems face considerable challenges in ensuring proper protein folding, accurate chain assembly, and the soluble expression of full-length immunoglobulins. Recent optimization strategies have focused on host engineering (e.g., modulating secretion pathways and chaperone proteins), the coordinated regulation of expression elements (e.g., optimizing the light-to-heavy chain ratio), and regulation of fermentation processes. In addition to summarizing the above strategies, this review discusses the progress made in expressing both full-length immunoglobulins and antibody fragments across different microbial hosts, analyzes the advantages and limitations of each system, and explores potential future directions, aiming to provide a reference for the efficient heterologous expression of immunoglobulins. Full article

Proteins are the most common targets for antibody discovery and vaccine development, but their sequence variability can limit the breadth of resulting antigens. Lipids represent an alternative class of antigens due to their structural conservation and roles in host–pathogen interactions. Here, we describe the development and optimization of an in vitro antibody selection workflow using lipid-containing nanodiscs as antigen presentation platforms to enable phage and yeast display selections under conditions adapted for these non-protein targets. Lipopolysaccharide (LPS) nanodiscs were first used as a model system to evaluate selection strategies, including competitive and subtractive approaches to reduce non-specific binders, yielding peptide and single-chain variable fragment (scFv) binders that were affinity matured to improve binding signals. The same approach was subsequently used to select scFv antibodies that recognize lipid nanodiscs prepared from Yersinia pestis membrane lipid extracts. These antibodies show binding to lipid nanodiscs derived from Y. pestis, with evidence of selectivity relative to control nanodiscs. Overall, this work establishes a workflow for antibody selection against lipid-containing nanodisc antigens and highlights practical considerations associated with these targets. The approach may be useful for generating affinity reagents to membrane-associated lipids, although further characterization is required to define antigen specificity and functional activity. Full article

A therapeutic antibody, CSL305, has been developed, which combines inhibition of the complement classical and lectin pathways via complement C2 binding with an ability to act as an antagonist of the neonatal Fc receptor (FcRn). CSL305 binds to human C2 (huC2) but shows no binding or activity against mouse C2 precluding its use in mouse models of disease to fully assess in vivo efficacy. To circumvent this, a mouse strain was developed that replaced the expression of mouse C2 with huC2 by homologous recombination. These mice (huC2 “knock-in”; KI) were shown to express huC2 protein and to have complement activity. Interestingly, male huC2-KI mice showed much stronger complement activity compared to female mice and were also sensitive to inhibition by CSL305. Two models of disease using male huC2-KI mice were then used to assess the in vivo efficacy of CSL305. The first was an attenuated passive anti-glomerular basement membrane (GBM) glomerulonephritis model involving complement activation as its primary mechanism of action. CSL305 showed dose-dependent inhibition of disease as measured by urine albumin, with reductions in kidney cellular infiltration and plasma C3 cleaved fragments C3b/C3c/iC3b also observed. The second model was a collagen autoantibody-induced arthritis (CAIA) mouse model. Here, CSL305 showed a significant and dose-dependent inhibition of clinical score in both prophylactic and therapeutic settings, mediated exclusively via its FcRn mechanism of action. Although the animal models used in this study were found to preclude the demonstration of a synergistic effect on both mechanisms, CSL305 does act in vivo as both a complement inhibitor and as a FcRn antagonist to ameliorate disease. Full article

This study aimed to determine the prevalence and risk factors associated with Toxoplasma gondii infection in blood donors from the Brazilian Semiarid region, and to explore its implications for transfusion safety. Samples were collected from 646 donors at blood donation centers in the states of Ceará and Paraíba. Serological diagnosis was performed using BIOLISA TOXOPLASMOSE ELISA kits for anti-T. gondii IgM and IgG antibodies, and molecular diagnosis was conducted by conventional PCR targeting a 529-bp noncoding repetitive fragment. Epidemiological questionnaires on variables associated with infection were administered, and statistical analysis was performed in univariate and multivariate stages, using multiple logistic regression. Among the 646 donors, 43.4% (281/646) were positive for anti-T. gondii IgG antibodies, 0.3% (2/646) for IgM antibodies, and none tested positive by PCR. In the univariate analysis, age, family income, educational level, salad washing practices, water source, raw milk consumption, and duration as a donor were significantly associated, whereas in the multivariate analysis only “age” and “salad washing practices” remained significant. A substantial IgG seroprevalence was observed among blood donors in the Brazilian Semiarid. The low IgM frequency, concurrent IgG positivity, and negative PCR results are consistent with a low transfusion risk in the region. However, these findings should be interpreted cautiously, as negative PCR results do not completely rule out the presence of circulating parasites. Age was identified as a risk factor, whereas proper salad washing showed a protective effect. Full article

Monoclonal antibodies (mAbs) have been the subject of extensive study in recent years due to their recognition as highly promising therapeutic molecules offering high specificity and a low risk of side effects. Monitoring the structure of these molecules is crucial for developing new therapeutics, characterizing interactions with antigens or receptors, and explaining potential changes in activity between antibody production batches. However, commonly used biophysical approaches provide only low-spatial-resolution information, and conventional structural biology techniques such as crystallography and cryo-electron microscopy (cryo-EM) are difficult to apply to these highly dynamic proteins. Solution nuclear magnetic resonance (NMR) spectroscopy is the method of choice for structural studies of flexible proteins at atomic resolution; however, it has traditionally been limited to low-molecular-weight biological systems. In this review, we present recent advances in NMR spectroscopy and advanced isotopic labeling methods that have enabled the atomic-resolution study of both the crystallizable (Fc) and antigen-binding (Fab) fragments of antibodies. We show how NMR is becoming a powerful tool for investigating full-length mAbs at an atomic level, opening up new possibilities for the characterization and in-depth quality control of therapeutic antibodies in solution. Full article

Single-domain antibodies (sdAbs) are the smallest antigen-binding antibody (Ab) fragments (12–15 kDa) and have emerged as a versatile therapeutic platform. Their compact size, high solubility, stability, and ability to access cryptic epitopes distinguish them from conventional monoclonal Abs (mAbs) and larger Ab fragments. These properties are particularly attractive in ophthalmology, where molecular size, tissue penetration, and formulation constraints critically influence therapeutic performance. This narrative review summarizes the structural features, engineering strategies, immunogenicity considerations, and production platforms of sdAbs, with a focus on ocular applications. Preclinical studies demonstrate promising efficacy in retinal vascular diseases through targeting of VEGFA, ANG2, TNFα, and complement components, as well as in inflammatory and anterior segment disorders. SdAbs can be formatted as multimeric or Fc-fused constructs to extend intraocular half-life or delivered via gene therapy vectors as a sustained intraocular “biofactory” approach. Notably, recent work demonstrates the feasibility of vector-encoded sdAbs targeting complement C3 in vivo. While challenges remain regarding immunogenicity, pharmacokinetics, and regulatory pathways, the approval of several sdAb-based drugs in other fields underscores their clinical potential. SdAbs represent a promising next-generation modality for ocular therapeutics, enabling innovative strategies beyond conventional antibody formats. Full article

Background/Objectives: Colistin (COL), administered as a prodrug colistimethate sodium (CMS), is commonly used to treat infections caused by multidrug-resistant Gram-negative bacteria in critically ill patients. Given high CMS instability, very complex and variable pharmacokinetics (PK) and high incidence of toxicity, therapeutic drug monitoring (TDM) of active COL might play an important role. This study aimed to develop and validate an accessible immunoassay-based approach for COL monitoring in human serum. Methods: A direct competitive enzyme-linked immunosorbent assay (dcELISA) was developed using polyclonal (pAb) anti-polymyxin antibody alongside a polymyxin B–horseradish peroxidase conjugate. CMS conversion to COL along with serum deproteinization was achieved using 5% trichloroacetic acid (TCA) treatment at 37 °C. Assay accuracy and precision were assessed by spike-and-recovery experiments in healthy volunteer serum. The assay was applied to serum samples from critically ill patients with burns or pneumonia receiving CMS therapy. The reliability of the measurements was confirmed by parallel dcELISA based on a reference monoclonal antibody (mAb) against fragmented polymyxin molecule. Results: Both ELISA formats demonstrated high sensitivity, with limits of detection of 0.053 ng/mL (pAb) and 0.047 ng/mL (mAb). TCA treatment achieved maximal CMS hydrolysis under tested conditions within one hour. Clinical sample analysis showed excellent agreement between the two assays (R² = 0.996), with Bland–Altman analysis revealing a minimal bias of 3.7%. Exploratory PK analysis in burn patients demonstrated increased total drug volume of distribution (45.7–64.9 L) and clearance (8.3–16.3 L/h). Conclusions: This is the first report of ELISA for COL TDM in critically ill patients. The method offers acceptable analytical performance and practical simplicity, with potential to broaden TDM access beyond specialist centers. Full article

Background: The monkeypox virus (MPXV) has attracted considerable global attention due to its potential to cause widespread outbreaks, necessitating the development of rapid and accurate diagnostic methods of significant clinical importance. A29, a key envelope protein of MPXV, represents a promising diagnostic target. Methods: A novel monoclonal antibody, D10, was isolated from the human Tomlinson I+J phage display library by biopanning against the recombinant A29 protein. The D10 Fab fragment was expressed and purified, and its binding affinity was characterized by biolayer interferometry. Molecular docking was performed to predict potential interacting residues. Specificity and detection performance were evaluated by direct and competitive enzyme-linked immunosorbent assay (ELISA). Results: D10 possesses a unique complementarity-determining region sequence and exhibits strong binding affinity toward the A29 protein. Structural modeling analysis suggested potential interacting residues of A29, including Gln67, Arg74, Asn75, Arg81, and Asn84, which may primarily interact with Ser10, Thr5, Gly49, Gly47, and Glu97 in the heavy chain of D10. The binding affinity, determined by biolayer interferometry, showed a dissociation equilibrium constant of 6.44 nM, indicating strong binding capability. Furthermore, competitive ELISA demonstrated that D10 binds selectively to the A29 protein, with a half-maximal inhibitory concentration of 1.88 μg/mL and a limit of detection of 0.12 μg/mL. Conclusions: Overall, this monoclonal antibody provides a valuable tool for the immunological detection of MPXV and holds potential for future clinical diagnostic applications. Full article

In this study, we combined computational predictions with experimental validation as a hybrid strategy to explore whether the E protein of tick-borne encephalitis virus (TBEV) possesses autoimmune potential. Using in silico homology searches, we identified two viral epitopes (evglekl and vtgtqgt) within the TBEV E protein that share sequence identity with fragments of the human proteins DNAH7 and CSMD2. Antibodies against these epitopes were detected in the plasma of a subset of patients after natural TBEV infection. Notably, no such antibodies were found in recipients of the Tick-E-Vac vaccine, indicating that the current vaccine does not induce cross-reactive humoral responses to these epitopes. Further computational analysis predicted that these epitopes could be presented by HLA class II molecules (alleles DRB1*09:01 and DRB1*07:01), which are known to be associated with autoimmune pathologies. Molecular dynamics simulations confirmed stable binding of the peptides within the HLA grooves, with favorable binding energies. These findings suggest a possible involvement of T-helper cells in the autoreactive process. Natural TBEV infection can give rise to antibodies against epitopes homologous to human proteins, particularly in genetically predisposed hosts. While such homology alone does not predict the onset of autoimmune disease, it represents a risk factor. Full article

Vector-borne protozoal infections—including babesiosis, theileriosis, hepatozoonosis, trypanosomosis, and leishmaniosis—impose a substantial burden on livestock and companion animal health worldwide and carry important zoonotic and public health implications. Accurate diagnosis is essential yet challenging, given the diversity of parasite genera, their markedly different tissue tropisms, and the uneven distribution of diagnostic resources across veterinary settings. This review provides an integrated overview of the principal diagnostic approaches available, structured around the biological logic that guides test selection in practice. Microscopic examination remains the first-line method; its strengths and limitations are discussed for intraerythrocytic parasites (Plasmodium spp., Babesia spp., Theileria spp., Cytauxzoon spp.—the latter two with additional extra-erythrocytic schizont stages in leukocytes and tissue macrophages, respectively), leukocyte-associated forms (Hepatozoon spp.), extracellular trypanosomes, and tissue-stage parasites, including emerging applications of artificial intelligence. Serological methods—enzyme-linked immunosorbent assay (ELISA), indirect fluorescence antibody test (IFAT), and point-of-care lateral flow assays—are evaluated for their role in exposure detection, population screening, and international trade certification, with attention to cross-reactivity and the active-versus-past-infection distinction. Molecular diagnostics, encompassing conventional PCR, qPCR, droplet digital PCR, isothermal amplification, and next-generation sequencing, are reviewed with respect to target selection, sensitivity, and point-of-care applicability. Finally, diagnostic challenges are contextualised within a One Health framework, highlighting the fragmentation of veterinary surveillance and the need for integrated, cross-sector approaches to detect emerging threats. Full article

This study aimed to express the canine parvovirus (CPV) VP2 protein prokaryotically and develop an indirect ELISA for detecting CPV-specific antibodies in canine serum. The VP2 gene from a laboratory-isolated CPV strain was amplified and cloned into the pET-28a vector. Following prokaryotic expression optimization, the recombinant protein was purified via Ni-NTA affinity chromatography and validated using Western blotting. An indirect ELISA was established utilizing the purified VP2 as the coating antigen, with optimal parameters determined by checkerboard titration. A 1773 bp VP2 fragment was amplified. Optimal expression of the 64.8 kDa recombinant VP2 was achieved with 2 mmol/L isopropyl β-D-thiogalactoside (IPTG) at 32 °C for 8 h. For the indirect ELISA, the optimal antigen coating concentration was 2 μg/mL, alongside primary (canine serum) and secondary antibody dilutions of 1:320 and 1:4000, respectively. The diagnostic cut-off optical density at 450 nm (OD₄₅₀) threshold was established at ≥0.2066, and the analytical sensitivity reached a serum dilution of 1:5120. Compared with the hemagglutination inhibition (HI) assay using 192 clinical serum samples, the ELISA showed a diagnostic sensitivity of 85.94%, a diagnostic specificity of 88.28%, and an overall agreement rate of 87.50%. The mean intra-assay and inter-assay coefficients of variation were 4.39% and 3.02%, respectively. These findings indicate that the recombinant VP2-based indirect ELISA showed good analytical sensitivity, reproducibility, and diagnostic agreement with the HI assay for detecting CPV-specific antibodies in canine serum under the tested conditions, although broader cross-reactivity validation is still required. Full article

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