Protein therapeutics are extensively used in the treatment of autoimmune diseases, but a subset of patients appears to be refractory to these treatments, mainly due to the development of an immune response to the drug. A better understanding of the mechanism underlying the therapeutic drug’s failure becomes fundamental for the development of new and more effective treatments. Unfortunately, there are few cases where the exact mechanisms through which drugs bypass immunological tolerance and provoke immunogenicity have been studied. In this context, peptide epitope identification gained increasing importance in investigating the molecular mechanism of therapeutic drug’s immune responses. Despite peptide identification and use to monitor anti-drug antibody (ADA) profiles is a promising research field, their use is far away from a wide application both at the research and at the commercial level. Herein it is reported a compilation of studies in which peptides are directly involved in anti-drug immune responses, becoming the molecular key step for a better understanding of refractory reactions in therapeutic drugs. An overview on T-cell and B-cell peptide recognition is given, showing the growing potential and advantages of peptides when used in the field of refractoriness to drugs. This review includes studies describing antigenic peptides that enable enhanced ADA detection directly in patients’ sera, as well as the proof of concept that asses the use of peptides instead of proteins, to facilitate the identification of neutralizing ADA.

The use of adalimumab biosimilars has become increasingly common in clinical practice, reflecting their growing acceptance and efficacy as therapeutic alternatives to reference biologics. However, studies investigating the molecular interactions between anti-adalimumab antibodies (AAA) elicited in patients and different adalimumab biosimilars remain limited. This study aims to characterize the kinetic interactions between purified AAA from pediatric patients with Juvenile Idiopathic Arthritis and three adalimumab formulations: the originator Humira®, and the biosimilars GP2017 (Hyrimoz®) and SB5 (Imraldi®). At this purpose, adalimumab formulations were immobilized on a biosensor, and purified AAA were flowed to perform further kinetic analysis using the surface plasmon resonance (SPR) technology. Results showed that the KD values for purified AAA from patients treated with biosimilars GP2017 or SB5 were comparable across all formulations tested, including the originator Humira®. AAA interacted with Humira®, Hyrimoz®, and Imraldi® with similar apparent affinity (10E-9 M > KD > 10E-10 M), slight variations have been observed among patients, less among biosimilars. The similarity in KD values across the biosimilars and the originator supports the notion that, at the level of immunogenicity, the biosimilars can be considered clinically comparable to the originator

Introduction: Calcium/calmodulin‐dependent protein kinase kinase 1 (CAMKK1) regulates energy homeostasis through AMP‐activated protein kinase (AMPK). CAMKK1 has been implicated in appetite and satiety regulation; however, its role in obesity or type 2 diabetes mellitus (T2DM) remains unexplored. In this cross‐sectional study, the primary aim was to confirm whether CAMKK1 is elevated in individuals with diabetes. The secondary aim was to investigate CAMKK1's molecular correlates. Methods: CAMKK1 serum levels in individuals with obesity ( n = 3,061), patients with T2DM ( n = 4,910) and controls ( n = 44,257) were retrieved and compared (age, body mass index—BMI and sex‐adjusted ANCOVA). Pearson correlation coefficients and linear regression coefficients (age and BMI‐adjusted) were computed. The moderation effect of diagnostic groups was also assessed. The interaction between factors was explored by mixed graphical models. Results: CAMKK1 was elevated in patients with T2DM, in comparison to both individuals with obesity and controls (post hoc comparison, Tukey‐adjusted p = 0.010 and p = 0.044, respectively). Across diagnostic groups, positive associations were observed between CAMKK1 and AMPK (min β > 0.400, max p < 0.001) or TNFα (min > β 0.070, max p < 0.001). A positive association with leptin ( β = 0.010, p = 0.002) and ghrelin ( β = 0.005, p = 0.048) was observed only within controls. Multivariate multivariable models confirmed that specific interactions between factors were disrupted in patients with T2DM ( p < 0.001). Conclusion: These findings provide new insights into the role of CAMKK1 in obesity and T2DM. Future research may further explore CAMKK1's interplay with inflammatory pathways.

The use of adalimumab biosimilars has become increasingly common in clinical practice, reflecting their growing acceptance and efficacy as therapeutic alternatives to reference biologics. However, studies investigating the molecular interactions between anti-adalimumab antibodies (AAA) elicited in patients and different adalimumab biosimilars remain limited. This study aims to characterize the kinetic interactions between purified AAA from pediatric patients with Juvenile Idiopathic Arthritis and three adalimumab formulations: the originator Humira^®, and the biosimilars GP2017 (Hyrimoz^®) and SB5 (Imraldi^®). For this purpose, adalimumab formulations were immobilized on a gold chip, and purified AAA were flowed to perform further kinetic analysis using the surface plasmon resonance (SPR) technology. Results showed that the K_D values for purified AAA from patients treated with biosimilars GP2017 (Hyrimoz^®) or SB5 (Imraldi^®) were comparable across all formulations tested, including the originator Humira^®. AAA interacted with Humira^®, Hyrimoz^®, and Imraldi^® with similar apparent affinity (10⁻⁹ M > K_D > 10⁻¹⁰ M); slight variations have been observed among patients, less among biosimilars. The similarity in K_D values across biosimilars and the originator supports the notion that, at the level of immunogenicity, biosimilars can be considered clinically comparable to the originator.

Membrane fusion between SARS-CoV-2 and host cells is mediated by the spike protein and involves the membrane-proximal external region (MPER), a tryptophan-rich sequence implicated in viral entry. Here, we investigated the feasibility of MPER-derived peptides as potential antiviral agents and examined the structural determinants underlying their activity. A series of truncated MPER peptides was synthesized and evaluated for antiviral activity against SARS-CoV-2 in cellular assays, revealing a potent inhibitory activity with nanomolar IC50 values associated to sequences rich in aromatic residues. Using NMR spectroscopy, we demonstrate that these peptides adopt a stable a-helical conformation in solution and membrane-mimetic environments that is stabilized by intramolecular aromatic π-π stacking interactions among tryptophan and tyrosine side chains. Structure–activity analysis indicates that this aromatic network promotes a-helix stabilization mimicking MPER structure of the spike post-fusion and correlates with enhanced antiviral potency. Our findings reveal a structural mechanism by which aromatic stacking stabilizes MPER helicity and drives antiviral activity, providing insights into peptide-based inhibition of viral membrane fusion and offering a framework for the rational design of SARS-CoV-2 entry inhibitors.

Background: SARS-CoV-2 fusion inhibitory peptides represent promising antiviral candidates. Recently, a 19-mer peptide (PN19)—designed in our laboratory to mimic the internal fusion peptide of the SARS-CoV-2 spike S2 subunit—demonstrated potent antiviral activity and stable conformational features. Objectives: To investigate how this antiviral activity depends on membrane interactions, we designed synthetic PN19 lipopeptide derivatives and evaluated their efficacy against SARS-CoV-2 replication. Methods: Lipopeptides were synthesized by conjugating cholesterol to either the N- or C-terminus of the PN19 peptide, utilizing a Gly/Ser pentapeptide (GSGSG) and/or various polyethylene glycol (PEG) spacers. Antiviral activity against SARS-CoV-2 variants was evaluated by plaque reduction assays, and cytotoxicity was assessed in Vero E6 cells. Results: The lipopeptides exhibited potent inhibitory activity at sub-micromolar concentrations. Compared to the unmodified PN19 peptide, antiviral efficacy was significantly enhanced by cholesterol conjugation at either terminus. Evaluation of six PN19 lipopeptides bearing the GSGSG sequence and different PEG spacers revealed that C-terminal cholesterol conjugation yielded higher antiviral activity than N-terminal derivatives. Furthermore, thirteen shorter PN19 lipopeptide derivatives (8–13-mers) confirmed this robust efficacy, which was most pronounced with C-terminal cholesterol conjugation and further enhanced by the spacers. Noteworthy, all tested PN19 lipopeptides displayed broad activity against multiple SARS-CoV-2 variants in the absence of cytotoxicity. Conclusions: Collectively, peptides conjugated with cholesterol at the C-terminus emerged as highly potent inhibitors of SARS-CoV-2, likely driven by enhanced peptide–membrane interactions. These findings warrant further investigation to fully elucidate the role of lipidation in the inhibitory mechanism, supporting the development of novel antiviral lipopeptides for SARS-CoV-2 therapy.