Background/Objectives: Cadherin-13 (CDH13), part of the cadherin family, is attached to the plasma membrane through glycosylphosphatidylinositol. CDH13 plays essential roles in the development of the neurological and vascular systems and is a risk factor for neural and cardiovascular diseases. CDH13 is expressed on the plasma membrane in both mature and uncleaved precursor forms with the prodomain. Although several anti-CDH13 monoclonal antibodies (mAbs) are available for basic research, there have been no reports of anti-CDH13 mAbs that can detect both the mature form and the uncleaved precursor in flow cytometry. Methods: We developed novel anti-human CDH13 mAbs (named Ca13Mabs) using the mature form of CDH13-expressed cells as an antigen. Results: Among Ca13Mabs, a clone, Ca13Mab-17 (IgG2b, κ) specifically recognized the mature and uncleaved precursor CDH13-overexpressed Chinese hamster ovary-K1 (CHO/CDH13) cells with no detectable cross-reactivity toward 21 other cadherins by flow cytometry. Ca13Mab-17 also detected endogenous CDH13 in human glioblastoma (LN229 and U87MG) and lung mesothelioma (NCI-H2052) cell lines. The dissociation constant (KD) value of Ca13Mab-17 for LN229 was estimated at 4.1 × 10−8 M. Furthermore, Ca13Mab-17 detected both the mature and uncleaved precursor CDH13 in Western blotting. It also identified new blood vessels and glioblastoma cells by immunohistochemistry. Conclusions: Ca13Mab-17 is a versatile tool for detecting both mature and uncleaved precursor forms of CDH13 and has potential for tumor diagnosis and therapy.

Background/Objectives: The immunoglobulin heavy-chain variable (IGHV) gene repertoire represents a characteristic feature of chronic lymphocytic leukemia (CLL), although its configuration is not well defined at the early disease stages. The IGHV repertoire of a cohort of early CLL patients was analyzed and compared to that of a “real-world” reference cohort. Methods: Patients from the O-CLL1 observational protocol, which enrolled only Binet stage A cases within twelve months from diagnosis, were studied. IGHV/IGHJ rearrangements were sequenced and annotated following ERIC recommendations, and stereotyped subsets were assigned using ARResT/AssignSubsets. The repertoire features were compared with the dataset of a real-world cohort of patients with heterogeneous staging (CTR cohort) and with published early-diagnosis series. Results: IGHV and IGHJ gene distributions and HCDR3-length profiles in O-CLL1 closely mirrored those of CTR, indicating that the BcR IG repertoire at diagnosis is already defined rather than being selected during disease progression. Mutated IGHV (M-CLL) predominated, with a frequency of stereotyped BcR IG comparable to that of other early-diagnosis cohorts. However, within this conserved framework, subset #4 was over-represented among M-CLL from O-CLL without an increased overall IGHV4-34 gene usage, suggestive of a selective expansion rather than a recombinational bias. Subset #4 cases retained canonical HCDR3 motifs and showed time-to-first-treatment like other M-CLL, likely reflecting the younger age structure of O-CLL1. Conclusions: Early-diagnosis CLL displays a biased IGHV repertoire with stereotyped configurations characteristic of CLL, including subsets that are rare in the normal B-cell repertoire. These findings support a central role for antigen-driven selection in shaping CLL evolution.

COVID-19 mRNA vaccines activate type I interferon pathways and in genetically or immunologically predisposed individuals may trigger autoimmune responses, including autoantibodies against melanoma differentiation-associated protein 5 (MDA5). Although cases of dermatomyositis (DM), particularly anti-MDA5-positive DM, have been increasingly reported after SARS-CoV-2 vaccination, its clinical spectrum and management remain incompletely defined. We conducted a narrative review of the literature on post-vaccination dermatomyositis, focusing on clinical features, autoantibody profiles, therapeutic approaches, and outcomes. The review was enriched by the inclusion of a new case: a 60-year-old woman who developed anti-MDA5-positive dermatomyositis two weeks after receiving her fourth dose of the BNT162b2 (Pfizer/BioNTech) vaccine. She presented predominantly with cutaneous and articular manifestations in the absence of interstitial lung disease. Treatment with oral prednisone, intravenous alprostadil, and the Janus kinase inhibitor tofacitinib resulted in marked clinical improvement. This case, together with the literature review, illustrates both typical and atypical presentations of vaccine-associated anti-MDA5 DM, highlights diagnostic challenges without lung involvement, and suggests JAK inhibition as a potential therapeutic option, contributing to a more comprehensive understanding of post-vaccination dermatomyositis.

Background: Anti-double-stranded DNA (anti-dsDNA) antibodies are a key serological marker for systemic lupus erythematosus (SLE) and are commonly assessed in conjunction with anti-nuclear antibody (ANA) testing by indirect immunofluorescence (IIF) on HEp-2 cells. However, their detection is influenced both by the heterogeneity of the autoimmune response and by the characteristics of the analytical method employed, thereby complicating diagnostic interpretation. Methods: In this retrospective single-center study, 3090 consecutive patients undergoing anti-dsDNA analysis were screened, and 138 positive individuals, with anti-dsDNA levels ≥ 15 IU/mL by fluoroenzyme immunoassay (FEIA), were included in the study. A control group of 29 anti-dsDNA-negative patients was also analyzed. Anti-dsDNA-positive patients were stratified by antibody level (low, mild, high), and the results were correlated with HEp-2 IIF titers and fluorescence patterns. Furthermore, in a subset of 30 positive patients, anti-dsDNA antibodies were evaluated using immunoblotting (IB) and the Crithidia luciliae indirect immunofluorescence test (CLIFT). Statistical analyses assessed associations and concordance among methods. Results: Higher anti-dsDNA levels were generally associated with higher HEp-2 IIF titers. However, a considerable percentage (35%) of patients with positive anti-dsDNA were negative by HEp-2 IIF. Notably, high anti-dsDNA levels were detected in 19% of HEp-2 IIF-negative patients (titer < 1:80), 18% of mildly HEp-2 IIF-positive patients (titer 1:80–1:160), and 25% of HEp-2 IIF-positive patients (titer > 1:320). In the subset of 30 positive patients, FEIA analysis showed high concordance with the immunoblot in both IIF-positive (81%) and -negative (100%) patients, while CLIFT demonstrated lower agreement with both FEIA and IB independently of the IIF. Conclusions: Our findings indicate that anti-dsDNA antibody detection may occur independently of HEp-2 IIF positivity and that FEIA, especially when confirmed by immunoblot, represents a reliable approach for anti-dsDNA assessment. The observed results in this study likely reflect differences in epitope recognition and assay sensitivity among methods, suggesting the use of a multi-step diagnostic strategy in the serological evaluation of SLE.

Background/Objectives: Developability assessment is a critical step in advancing antibody-based molecules toward clinical application. This evaluation typically begins during clinical candidate selection and continues throughout all modifications of the molecule during development. It is guided by the target product profile, which includes the intended administration route and regimen, formulation parameters, and process conditions encountered during manufacturing, storage, and delivery. While developability testing is well established for conventional therapeutic antibodies, strategies for assessing single-domain antibodies (sdAbs) and their conjugates remain underexplored. Here, we present a strategy to test the developability of sdAbs as a case study for two clinical candidates intended as precursors for the production of diagnostic tracers for clinical imaging. Methods: Assays were developed to evaluate chemical and thermodynamic stability, target binding affinity and capacity, and chelation efficiency (“chelatability”). Accelerated stability studies were conducted for both unconjugated sdAbs and their chelator conjugated forms following incubation at two pH conditions, at multiple time points, and after twelve freeze–thaw cycles to simulate process conditions and long-term storage. Analytical assays were applied stepwise in a hierarchical approach to minimize experimental effort and material consumption. Candidates exhibiting critical developability features were selectively addressed by assays with increasing precision. Results: A tailored panel of analytical assays optimized for low molecular weight proteins was established and applied to the two clinical candidates, identifying instability hotspots as well as potential mitigation strategies. Successful engineering of a candidate with an initially critical developability profile was achieved. Conclusions: This study demonstrates the implementation of a structured developability assessment strategy for sdAb conjugates. The approach integrates physicochemical and functional stability evaluations, supporting robust candidate selection, formulation development, and method optimization for this class of molecules.

Background/Objectives: Viral hepatitis A–E represents a significant public health problem. Data on the prevalence of viral hepatitis markers among alcoholics are inconsistent. Methods: The study included 151 patients treated for alcohol abuse in one Croatian center. The control group consisted of 110 individuals from the general population tested for a routine check-up. The prevalence of viral hepatitis markers was assessed using serology and molecular methods. Results: The prevalence rates of hepatitis markers among patients were as follows: anti-HAV, 15.2%; anti-HBs, 11.9%; anti-HBc/anti-HBs, 2.6%; anti-HCV, 4.0%; and anti-HEV, 14.6%. HCV RNA was detected in one patient (0.6%). Compared with the control group, patients showed significantly higher HCV seroprevalence (4.0 vs. 0%), while the prevalence of other hepatitis markers did not differ significantly between the groups. The anti-HAV prevalence was associated with age (from 0% in patients aged <40 years to 42.9% in patients aged 60+ years), employment status (highest among retired individuals at 46.2%), and age of occasional alcohol consumption (highest seroprevalence of 26.3% in those who reported consumption between 22 and 25 years). The association between anti-HEV and educational level was of borderline significance. Logistic regression showed that older and retired patients and those who consumed alcohol occasionally between 22 and 25 years showed higher odds for HAV seropositivity (OR = 11.454–49.400, OR = 6.857, and OR = 4.464, respectively). Patients with university degrees were at lower risk for HEV seroprevalence (OR = 0.083). Conclusions: Alcoholic patients showed a higher HCV seroprevalence than the general population, while the prevalence of other viral hepatitis markers did not differ between the groups. Further studies on a larger cohort of patients are needed to confirm these findings.

Background: Collagen XIα1, encoded by the COL11A1 gene, is a minor fibrillar collagen that is overexpressed in various human cancers, in which its presence correlates with tumor aggressiveness and progression. Methods: In this study, we developed two novel mouse monoclonal antibodies (mAbs)—anti-colXIα1 clone 3 and anti-colXIα1 clone 9—that target the putative C-telopeptide of human collagen XIα1. These antibodies target the RRHTEGMQA sequence, a unique nine-amino-acid stretch within the putative C-telopeptide of human collagen XIα1. Results: Corresponding to nearly identical V(D)J gene segments and complementarity-determining regions (CDRs), the antibodies specifically bound the RRHTEGMQA epitope in ELISAs but did not react with the C-propeptide. This specificity was further confirmed with the purified anti-colXIα1 clone 9 mAb, which demonstrated strong reactivity against recombinant proteins containing the RRHTEGMQA sequence in both ELISAs and Western blot assays. This sequence seems to behave as a linear B-cell neoepitope, in which the RRHT motif is crucial for epitope recognition. Otherwise, no immunodetections were observed, either in cultures and lysates from the COL11A1-highly expressing A204 human cell line or on tissue sections from specimens of human pancreatic ductal adenocarcinoma (PDAC), with strong desmoplastic reactions. Conclusions: Given the lack of precise knowledge of the characteristics of the putative C-telopeptide of human collagen XIα1, the presented antibodies could enhance our understanding of the processing of human procollagen XIα1 and contribute to better characterization of the tumor microenvironment of COL11A1-expressing cancers.

Background/Objectives: The SARS-CoV-2 virus frequently undergoes mutations to evade the human immune system. Vaccines for new strains are developed each season, and an identification test confirming the specific strain is essential for vaccine quality control, as stated by the U.S. Food and Drug Administration. However, a shorter timeline of antibody discovery was required to adjust vaccine development schedules. Therefore, anti-SARS-CoV-2 strain-specific, single-domain antibodies (sdAbs) for SARS-CoV-2 vaccines were discovered using alpaca synthetic libraries without animal immunization. Methods: A synthetic sdAb library was developed based on conserved alpaca sdAb frameworks, with a degree of freedom in the three complementarity-determining regions. Specific and high-affinity sdAb clones were selected from the library by one ribosomal display round, followed by two phage display selections using a biotinylated strain-specific SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as bait and non-biotinylated RBD variants to block. The sdAbs clones were applied to the identification test using Western blotting. The binding epitopes were determined by hydrogen–deuterium exchange mass spectrometry. Results: Five clones of XBB.1.5 and two clones of JN.1-specific sdAbs were discovered. Anti-JN.1 sdAb clone 1B9 detected JN.1 vaccine products but no other previously produced vaccine strains, Wuhan, BA.5 and XBB.1.5, by WB for vaccine identification test. Four binding epitopes for anti-JN.1 sdAb clone 1B9 were identified, including the L455S mutation, a critical amino acid to evade neutralizing antibodies for the JN.1 strain. Conclusions: Anti-XBB.1.5 and JN.1-specific sdAbs were discovered from a synthetic single-domain antibody library within 8–9 weeks, and these sdAbs were applied to vaccine identification testing.

Objectives: The epidermal growth factor receptor (EGFR) is a clinically relevant membrane receptor that is frequently overexpressed or dysregulated in multiple types of cancer, making it an important target for antibody-based strategies. Nanobodies, derived from camelid heavy-chain antibodies, possess favorable properties such as small size, high stability, and strong antigen-binding capacity. This study aimed to generate EGFR-specific nanobodies and to systematically characterize their binding properties and initial functional activity. Methodology: Bactrian camels were immunized with a whole-cell antigen prepared from 293F cells transiently transfected to express full-length human EGFR. A high-diversity phage display nanobody library was constructed from peripheral blood lymphocytes. After two rounds of biopanning against EGFR, positive clones were screened and selected. The identified nanobodies were recombinantly expressed in Escherichia coli and purified. Binding specificity, epitope relationships, and kinetic parameters were evaluated using high-performance liquid chromatography (HPLC), bio-layer interferometry (Octet), and flow cytometry. The effect of selected nanobodies on EGF-induced cell proliferation was evaluated using a CCK-8 assay. Results: Two EGFR-specific nanobodies, Nb2H4 and Nb2B6, were successfully isolated. Both nanobodies exhibited specific binding to EGFR and recognized distinct, non-competing epitopes. Kinetic analyses revealed favorable binding affinities, and flow cytometry confirmed their ability to recognize EGFR in its native cellular context. In addition, Nb2H4 significantly suppressed EGF-induced proliferation in an EGFR-overexpression cell model, indicating preliminary functional activity. Conclusions: This study reports on the successful generation and in vitro characterization of EGFR-targeting nanobodies based on the extracellular domain of EGFR. The identified nanobodies provide useful molecular tools for epitope mapping, structural studies, and the further exploration of EGFR-directed antibody engineering strategies.

Background: This study assesses the robustness of a legacy N-glycan profiling method for the therapeutic antibody MAB1 with different Peptide-N-glycosidase F (PNGase F) enzyme sources, solid phase extraction (SPE) cartridges, and reagent stability, aligning with ICH Q14 lifecycle management principles. Glycosylation profiling is critical for therapeutic antibodies as it influences both function and pharmacokinetics. Method: The legacy N-glycan profiling method, 2-aminobenzoic acid (2-AA) labeling combined with normal-phase HPLC, was re-evaluated to confirm consistent analytical performance in the context of evolving regulatory expectations. The evaluation focused on three key factors: PNGase F enzyme sources, solid-phase extraction (SPE) cartridges, and reagent stability. Results: Commercial PNGase F enzymes showed various performances, with some sources yielding significant differences. Several SPE cartridges were also tested, with certain formats displaying poor recovery and high variability, particularly for sialylated glycans. In addition, reagent stability studies revealed rapid degradation of the labeling reagent within a few days. Conclusions: These results underscore the importance of risk control, continual improvement, and lifecycle management to ensure reliable glycosylation analysis and the sustained robustness of legacy methods.