Introduction Immune checkpoint inhibitors (ICI) have revolutionized the treatment of metastatic malignant melanoma (MM) and improved long-term survival. Despite the impressive results, some patients still have progressive disease, and the search for biomarkers predicting response to ICI treatment is ongoing. In this search, galectin-3 (Gal-3) has been suggested as a molecule of interest, both as a marker of treatment response and as a treatment target to potentiate ICI therapy. We have previously demonstrated the binding between programmed cell death 1 (PD-1) and Gal-3, and here, we investigated the interaction between PD-1, pembrolizumab, and Gal-3 in metastatic MM patients. Methods The binding between PD-1, pembrolizumab and Gal-3 was investigated by surface plasmon resonance (SPR) and cryogenic electron microscopy (cryo-EM). The function was studied in in vitro cultures and soluble levels of both PD-1 and Gal-3 were measured in metastatic MM patients, treated with pembrolizumab. Results By SPR, we demonstrated that Gal-3 can block the binding between PD-1 and pembrolizumab, and further visualized a steric inhibition using cryo-EM. T cells cultured with Gal-3 had reduced pro-inflammatory cytokine production, which could not be rescued by pembrolizumab. In patients with metastatic MM, high levels of Gal-3 in plasma were found in patients with a longer progression-free survival in the study period, whereas high Gal-3 expression in the tumor was seen in patients with disease progression. Soluble PD-1 levels in plasma increased after treatment with pembrolizumab and correlated with disease progression. Conclusion We demonstrate that the interaction between PD-1 and Gal-3 interferes with the binding of pembrolizumab, supporting that an immune suppression induced by Gal-3 in the tumor microenvironment cannot be rescued by pembrolizumab.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and bone erosions. The glycosylated programmed death‐1 (PD‐1) receptor plays an important role in regulating immune responses and maintaining tolerance. In this study, we focus on two features observed in RA: impaired PD‐1 signalling and Galectin‐3 (Gal‐3) upregulation. We hypothesize that Gal‐3 binds PD‐1 and PD‐1 ligands, potentially contributing to impaired PD‐1 signalling. PD‐1 and Gal‐3 levels in RA synovial fluid (SF) and plasma were evaluated by ELISA. PD‐1 and Gal‐3 interaction was examined by Surface Plasmon Resonance and ELISA. PD‐1, PD‐L1 and Gal‐3 expression on mononuclear cells from SF and peripheral blood as well as fibroblast‐like synoviocytes were examined by flow cytometry. Effects of Gal‐3 and PD‐L1 on osteoclast formation was evaluated by tartrate‐resistant acid phosphatase assay. We show that Gal‐3 binds PD‐1 and PD‐L1. Results demonstrated high expression of PD‐1 and Gal‐3 on mononuclear cells, especially from SF. Gal‐3 inhibited PD‐1 signalling when PD‐L1 was present. Furthermore, a role of Gal‐3 in osteoclast formation was observed in vitro, both directly but also through PD‐1:PD‐L1 inhibition. Effects of Gal‐3 on the PD‐1 signalling axis are proposed to be inhibitory, meaning high Gal‐3 levels in the complex synovial microenvironment are not desirable in RA. Preventing Gal‐3's inhibitory role on PD‐1 signalling could, therefore, be a therapeutic target in RA by affecting inflammatory T cell responses and osteoclasts.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterised by heterogeneous clinical manifestations and varying degrees of organ involvement. The CD40‐CD40 Ligand (CD40L) pathway is implicated in autoimmune responses, with elevated levels of soluble CD40L (sCD40L) observed in SLE patients. This study investigates sCD40L as a biomarker for disease activity and its utility in stratifying patients for CD40L‐targeted therapies. Plasma levels of sCD40L were quantified in SLE patients (n = 169) and healthy controls (n = 100). Correlations between sCD40L levels and disease activity measures were analysed using Spearman's correlation and logistic regression. K‐means clustering grouped patients based on sCD40L concentrations, and principal component analysis (PCA) was performed to assess relationships among disease activity variables. SLE patients exhibited significantly higher sCD40L levels (median 2.2 ng/mL) than healthy controls (median 0.81 ng/mL; p = 0.0079). Elevated sCD40L levels correlated weakly with higher SLE Disease Activity Index (SLEDAI) scores (p = 0.0418), positive Anti‐Nuclear Antibody status (p = 0.0133), increased IgG levels (p = 0.0148) and decreased lymphocyte counts (p = 0.0327). Clustering analysis and PCA revealed that patients with higher sCD40L levels tended to have increased disease activity, elevated anti‐dsDNA antibody concentrations, and higher C‐reactive protein (CRP) levels. Elevated sCD40L levels in SLE patients correlate with disease activity markers, suggesting its potential as a biomarker for monitoring disease progression and severity. Additionally, sCD40L may aid in stratifying patients who could benefit from CD40L‐targeted therapies within a treat‐to‐target framework. Further longitudinal studies with larger cohorts are warranted to validate and explore the role of sCD40L in personalised SLE treatment strategies.

Dysregulation of the CD40‐CD40L axis is implicated in autoimmune diseases. Early clinical trials targeting CD40L with antibodies failed due to Fc‐mediated side effects. To address this, we developed an anti‐CD40L Fab fragment, Fab20, designed to block B‐cell activation. Fab20 was evaluated for its binding properties, CD40‐CD40L inhibition, and effects on human B‐cell activation and differentiation using immunoassays, cryo‐electron microscopy, flow cytometry, and cell cultures. Fab20 binds CD40L with a dissociation constant of 70 nM. Structural analysis revealed a “propeller‐like” structure consisting of three Fabs binding to the CD40L trimer, sterically blocking parts of the CD40 binding site. Fab20 effectively inhibited B‐cell activation, maintaining naïve B cells in their inactive state, and suppressed antibody (IgG) production over 14 days. Fab20 represents a promising novel therapeutic approach for treating autoimmune diseases driven by CD40‐CD40L dysregulation. Its mechanism of action, coupled with the absence of Fc‐mediated effects, suggests a favorable safety profile.

RNA therapeutics are powerful tools for gene modulation and targeted therapies, but their clinical application is hindered by nuclease degradation and immunogenicity. Incorporating chemical modifications, like locked nucleic acids (LNAs), can enhance nuclease resistance, targeting properties, and thermal stability. Traditionally, LNA incorporation has relied on solid‐phase synthesis of short RNAs. Engineered polymerases capable of incorporating xenonucleic acids (XNAs), including LNA, into longer RNAs have been described. However, their XNA yield is limited by primer and template copy numbers, and the generated DNA‐XNA duplexes can be difficult to purify. We present a novel approach for incorporating LNA‐ATP and LNA‐TTP alongside 2′fluoro (2′F)‐modified pyrimidines via in vitro transcription using a mutant T7 RNA polymerase. This method enables efficient, primer‐independent synthesis and amplification of LNA‐modified RNA with low error rates. To demonstrate its utility, we performed systematic evolution of ligands by exponential enrichment (SELEX) to select LNA‐ and 2′F‐modified aptamers targeting influenza hemagglutinin and human CD40 ligand, two therapeutically relevant proteins. Iterative SELEX cycles yielded aptamers with low‐nanomolar affinities, high specificity, increased thermal stability, and high nuclease resistance. Overall, this approach provides a scalable and versatile platform for generating chemically stabilized RNAs, fully compatible with SELEX, and holds potential for developing next‐generation RNA‐based therapeutics with improved pharmacokinetics.

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.