Antibodies

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: 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.