Background/Objectives: Mammalian display enables direct screening of full-length IgG antibodies, but genetically controlled library construction remains technically demanding. Here, we developed a mammalian display platform in which a target-naïve ΔDH antibody scaffold is first established at a defined human artificial chromosome (HAC) acceptor site, and CDR-H3 diversity is then introduced directly into that fixed genomic background by CRISPR-Cas9-mediated homology-directed repair (HDR). Methods: A site-specifically integrated display cell line carrying the ΔDH scaffold on an HAC was generated by Cre-mediated recombination. Randomized CDR-H3 libraries were introduced into this defined genomic background by HDR and screened by magnetic- and fluorescence-activated cell sorting together with orthogonal antigen-detection formats. Binding of isolated clones was characterized by flow cytometry and enzyme-linked immunosorbent assay. Results: Although early selection enriched clones reactive with a fluorescent reagent rather than the intended antigen, refinement of the selection scheme enabled isolation of AFFVVF. This selected antibody variant recognized multiple SARS-CoV-2 receptor-binding domain (RBD) variants by flow cytometry, with no detectable binding to unrelated antigens. As a recombinant protein, AFFVVF bound the JN.1 and XEC spike trimers by ELISA, but not monomeric RBD, including the XEC RBD used for selection, indicating that the form of antigen recognized differed between the cell-based and plate-based assays. Exploratory selections against epidermal growth factor receptor (EGFR) and glycoprotein A33 (GPA33) also produced antigen-dependent population shifts and enrichment of discrete sequence variants, although individual candidates were not independently validated as recombinant antibodies. Conclusions: These results provide proof of principle that targeted CDR-H3 diversification within a pre-established, genetically defined, target-naïve full-length IgG scaffold can yield a new antigen-binding antibody without starting from a pre-existing antigen-specific antibody or repertoire.
Background/Objectives: Regulatory rheumatoid factor (regRF) is a recently discovered population of natural anti-idiotypic antibodies carrying a shared paratope specific to the PD-1 molecule. Normally, regRF exerts a cytotoxic effect on PD-1-expressing T lymphocytes, including autoreactive ones, thereby controlling their expansion. The specificity for PD-1 indentifies regRF as a promising therapeutic agent for suppressing autoimmune reactions via the PD-1 pathway. The aim of this study was to develop gentle methods for regRF isolation from healthy blood plasma, which would preserve the bioactivity of regRF. Methods: Ammonium sulfate precipitation was used for crude regRF purification. A search for suitable affinity sorbents was carried out. The purified regRF was tested for its bioactivity. Results: Ammonium sulfate precipitation at 35% saturation, followed by extraction with 28% saturated ammonium sulfate solution, allowed the separation of regRF from a portion of non-target IgG and high-molecular-weight fibrinogen. RegRF showed high affinity for Cibacron Blue and no binding to Protein A. Sequential affinity chromatography on immobilized Cibacron Blue and Protein A enabled the purification of regRF from non-target immunoglobulins and low-molecular-weight fibrinogen. The purified regRF samples showed binding to PD-1 and reduced the PD-1+CD4+ T lymphocyte count in vitro. Conclusions: Thus, we have identified sorbents and conditions for the purification of biologically active regRF.
Background/Objectives: Blood count-derived inflammatory indices have been proposed as accessible biomarkers of systemic inflammation in psoriasis, but their longitudinal behavior during selective interleukin-23 blockade remains uncertain. We evaluated these indices in psoriasis patients treated with risankizumab. Methods: We conducted a multicenter retrospective real-world study (DERMIS), including adults with cutaneous psoriasis, with or without psoriatic arthritis, treated with risankizumab across 10 Italian centers, from national reimbursement up to 1 December 2025. Psoriasis Area and Severity Index (PASI) and complete blood count-derived indices—neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), systemic immune-inflammation index (SII), and systemic inflammation response index (SIRI)—were assessed at baseline and at weeks 12, 24, 52, 104, 156, and 208. Analyses used available data and sensitivity cohorts with follow-up at weeks 52, 104, and 208. Results: Among 250 patients, 43 had data at week 208. PASI scores decreased significantly at all follow-up time points. In contrast, systemic indices showed heterogeneous, non-linear trajectories: NLR, SII, and SIRI fluctuated without consistent sustained reductions, whereas MLR remained broadly stable. PLR showed the most reproducible downward trend, with nominal reductions at weeks 24 and 52; after Bonferroni correction across the six repeated visits within PLR, the reduction at week 52 remained statistically significant, whereas the week-24 reduction did not. Body mass index (BMI)-related differences were inconsistent and did not remain significant after Bonferroni correction. Sex, cardiometabolic comorbidities, and previous biological exposure showed no consistent effect. Conclusions: These findings challenge the use of routine blood count-derived indices as reliable biomarkers of cutaneous response to risankizumab.
Background/Objectives: Rituximab is widely used in primary glomerular diseases, but treatment may fail for several reasons, including primary non-response, incomplete response, relapse, intolerance or anti-rituximab antibodies. We assessed within-patient efficacy and safety after switching to another anti-CD20 antibody, considering the reason for switching. Methods: PubMed and Scopus were searched from inception to 15 May 2026 without language restrictions. Eligible reports included adults with primary glomerular disease treated with a non-rituximab anti-CD20 antibody after rituximab and reporting within-patient outcomes. Risk of bias was assessed with design-specific JBI tools and certainty with GRADE. Heterogeneity precluded meta-analysis, and findings were synthesized narratively. Results: A total of 309 patients were included across 43 studies: 283 had membranous nephropathy (MN), 18 had minimal change disease (MCD), 6 had focal segmental glomerulosclerosis (FSGS), and 2 had IgA nephropathy (IgAN). Obinutuzumab was the principal drug used, and the main reasons for switching were primary non-response, with immunization, relapse, or intolerance occurring less frequently. In MN, among the 262 patients with available response data, 217 achieved complete or partial remission, and relapse was rare. For MCD, 17 of 18 patients with active disease attained remission, and only one relapsed during follow-up. On the other hand, five out of the six FSGS patients responded, but three of them relapsed. The two patients with IgAN achieved either complete or partial remission. Notably, these results may not apply to conventional IgAN, since one biopsy showed contextually MCD and the other was proliferative and crescentic. Infusion-related reactions were the most frequent adverse events and were generally mild, while infections were the main issue during the follow-up. Evidence certainty for all outcomes was very low. Conclusions: Switching to a non-rituximab anti-CD20 antibody may be a feasible rescue strategy, most convincingly after intolerance, rituximab immunization, or relapse. The uncontrolled, very-low-certainty evidence precludes firm recommendations, and controlled studies are warranted.
Antibodies in ocular disease have heterogeneous biological meanings: they may act as direct pathogenic effectors, systemic diagnostic biomarkers, indicators of B-cell clonality, or therapeutic agents. This distinction is particularly important in the eye, where immune privilege and the blood–ocular barrier restrict tissue access, whereas inflammation may permit entry of immunoglobulins, complement, and immune cells or support local humoral responses. This narrative review integrates antibody and B-cell biology across autoimmune and paraneoplastic retinopathies, aquaporin-4-immunoglobulin G-positive neuromyelitis optica spectrum disorder, myelin oligodendrocyte glycoprotein antibody-associated disease, ocular mucous membrane pemphigoid, ocular myasthenia gravis, thyroid eye disease, systemic autoimmune and fibroinflammatory disorders with ocular involvement, and B-cell malignancies including vitreoretinal and ocular adnexal lymphoma. We distinguish the evidence for direct antibody pathogenicity from disease association and emphasize that a positive result is interpretable only in relation to the ocular phenotype, assay platform, titer, specimen compartment, and orthogonal confirmation. Serum testing, ocular-fluid analysis, electrophysiology, multimodal imaging, cytology, histopathology, immunophenotyping, immunoglobulin clonality, and molecular testing have complementary roles. Therapeutic strategies include B-cell or B-lineage depletion, complement inhibition, cytokine- or receptor-directed monoclonal antibodies, insulin-like growth factor-1 receptor blockade, and emerging FcRn- or plasma-cell-directed approaches; however, regulatory status and ocular-specific evidence vary substantially among diseases. A mechanistically stratified framework may reduce overdiagnosis, improve recognition of malignant masquerade syndromes, and support more precise selection of targeted therapy.
