Antibodies最新文献

Background: Bispecific antibodies represent a promising class of biologics for cancer treatment. However, their dual specificity and complex structure pose challenges in the engineering process, often resulting in molecules with good functional but poor physicochemical properties. Method: To overcome limitations in the properties of an anti-5T4 x anti-CD3 (α5T4 x αCD3) DART molecule, a phage-display method was developed, which succeeded in simultaneously engineering cross-reactivity to the cynomolgus 5T4 ortholog, improving thermostability and the elevating expression level. Results: This approach generated multiple DART molecules that exhibited significant improvements in all three properties. The lead DART molecule demonstrated potent in vitro and in vivo anti-tumor activity. Although its clearance in human FcRn-transgenic mice was comparable to that of the parental molecule, faster clearance was observed in cynomolgus monkeys. The lead α5T4 x αCD3 DART molecule displayed no evidence of off-target binding or polyspecificity, suggesting that the increased affinity for the target may account for its accelerated clearance in cynomolgus monkeys. Conclusions: This may reflect target-mediated drug disposition (TMDD), a potential limitation of targeting 5T4, despite its limited expression in healthy tissues.

The elicitation of broadly neutralizing antibodies (bnAbs) is a major goal of vaccine design for highly mutable pathogens, such as influenza, HIV, and coronavirus. Although many rational vaccine design strategies for eliciting bnAbs have been devised, their efficacies need to be evaluated in preclinical animal models and in clinical trials. To improve outcomes for such vaccines, it would be useful to develop methods that can predict vaccine efficacies against arbitrary pathogen variants. As a step in this direction, here, we describe a simple biologically motivated model of antibody reactivity elicited by nanoparticle-based vaccines using only antigen amino acid sequences, parametrized with a small sample of experimental antibody binding data from influenza or SARS-CoV-2 nanoparticle vaccinations. Results: The model is able to recapitulate the experimental data to within experimental uncertainty, is relatively insensitive to the choice of the parametrization/training set, and provides qualitative predictions about the antigenic epitopes exploited by the vaccine, which are testable by experiment. For the mosaic nanoparticle vaccines considered here, model results suggest indirectly that the sera obtained from vaccinated mice contain bnAbs, rather than simply different strain-specific Abs. Although the present model was motivated by nanoparticle vaccines, we also apply it to a mutlivalent mRNA flu vaccination study, and demonstrate good recapitulation of experimental results. This suggests that the model formalism is, in principle, sufficiently flexible to accommodate different vaccination strategies. Finally, we show how the model could be used to rank the efficacies of vaccines with different antigen compositions. Conclusions: Overall, this study suggests that simple models of vaccine efficacy parametrized with modest amounts of experimental data could be used to compare the effectiveness of designed vaccines.

Background/objectives: Photoimmunotherapy (PIT) is an innovative approach for the targeted therapy of cancer. In PIT, photosensitizer dyes are conjugated to tumor-specific antibodies for targeted delivery into cancer cells. Upon irradiation with visible light, the photosensitizer dye is activated and induces cancer-specific cell death. In the present article, we describe the PIT of prostate cancer (PC) as a therapeutic option for the targeted treatment of localized prostate cancer.

Methods: We conjugated the silicon phthalocyanine dye WB692-CB2 to recombinant cysteine-modified anti-CD44 and anti-EpCAM antibodies via a maleimide linker and tested the antibody dye conjugates for PIT on PC cells and prostate cancer stem cell (PCSC)-like cells.

Results: The anti-CD44 and anti-EpCAM antibody dye conjugates showed specific binding and high cytotoxicity against PC and PCSC-like cells following irradiation with red light. Combined treatment with both conjugates led to enhanced cytotoxic effects.

Conclusions: PIT with our dye WB692-CB2 can serve as an effective focal therapy against prostate cancer, preserving the prostate gland and minimizing side effects. It can be employed during radical prostatectomy (RP) to treat residual tumor cells or lymph node metastases in areas where further surgical intervention is not feasible. Utilizing multiple conjugates against antigens expressed on differentiated PC and PCSC-like cells, such as CD44 and EpCAM, could be an effective method to eradicate residual cancer cells in heterogeneous tumors. This approach could reduce the risk of local recurrence after RP and thus increase the therapeutic outcome of PC patients.

This review describes mass spectrometry (MS)-based approaches for the absolute quantification of therapeutic monoclonal antibodies (mAbs), focusing on technical challenges in sample treatment and calibration. Therapeutic mAbs are crucial for treating cancer and inflammatory, infectious, and autoimmune diseases. We trace their development from hybridoma technology and the first murine mAbs in 1975 to today's chimeric and fully human mAbs. With increasing commercial relevance, the absolute quantification of mAbs, traceable to an international standard system of units (SI units), has attracted attention from science, industry, and national metrology institutes (NMIs). Quantification of proteotypic peptides after enzymatic digestion using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has emerged as the most viable strategy, though methods targeting intact mAbs are still being explored. We review peptide-based quantification, focusing on critical experimental steps like denaturation, reduction, alkylation, choice of digestion enzyme, and selection of signature peptides. Challenges in amino acid analysis (AAA) for quantifying pure mAbs and peptide calibrators, along with software tools for targeted MS data analysis, are also discussed. Short explanations within each chapter provide newcomers with an overview of the field's challenges. We conclude that, despite recent progress, further efforts are needed to overcome the many technical hurdles along the quantification workflow and discuss the prospects of developing standardized protocols and certified reference materials (CRMs) for this goal. We also suggest future applications of newer technologies for absolute mAb quantification.

Recent avian influenza outbreaks have heightened global concern over viral threats with the potential to significantly impact human health. Influenza is particularly alarming due to its history of causing pandemics and zoonotic reservoirs. In response, significant progress has been made toward the development of universal influenza vaccines, largely driven by the discovery of broadly neutralising antibodies (bnAbs), which have the potential to neutralise a broad range of influenza viruses, extending beyond the traditional strain-specific response. This could lead to longer-lasting immunity, reducing the need for seasonal vaccinations, and improve preparedness for future pandemics. This review offers a comprehensive analysis of these antibodies, their application in clinical studies, and both their potential and possible shortcomings in managing future influenza outbreaks.

Background: The prediction of human clearance (CL) and subcutaneous (SC) bioavailability is a critical aspect of monoclonal antibody (mAb) selection for clinical development. While monkeys are a well-accepted model for predicting human CL, other preclinical species have been less-thoroughly explored. Unlike CL, predicting the bioavailability of SC administered mAbs in humans remains challenging as contributing factors are not well understood, and preclinical models have not been systematically evaluated.

Methods: Non-clinical and clinical pharmacokinetic (PK) parameters were mined from public and internal sources for rats, cynomolgus monkeys, and humans. Intravenous (IV) and SC PK was determined in Sprague Dawley rats for fourteen mAbs without existing PK data. Together, we obtained cross-species data for 25 mAbs to evaluate CL and SC bioavailability relationships among rats, monkeys, and humans.

Results: Rat and monkey CL significantly correlated with human CL and supported the use of species-specific exponents for body-weight-based allometric scaling. Notably, rat SC bioavailability significantly correlated with human SC bioavailability, while monkey SC bioavailability did not. Bioavailability also correlated with clearance.

Conclusions: The rat model enables an early assessment of mAb PK properties, allowing discrimination among molecules in the discovery pipeline and prediction of human PK. Importantly, rat SC bioavailability significantly correlated with human SC bioavailability, which has not been observed with other species. Rats are cost-effective and efficient relative to monkeys and provide a valuable tool for pharmacokinetic predictions in therapeutic antibody discovery.

Background/Objectives: Anorexia nervosa (AN) is a complex psychiatric disorder characterized by an extreme fear of gaining weight, leading to severe calorie restriction and weight loss. Beyond its psychiatric challenges, AN has significant physical consequences affecting multiple organ systems. Recent research has increasingly focused on the interplay between autoantibodies, oxidative stress, and nutritional state in this condition. Methods: Ninety-six subjects were evaluated: forty-eight with AN and forty-eight normal-weight control subjects. The serum levels of IgG reactive to hypothalamic antigens, uric acid, and total antioxidant capacity were evaluated by laboratory assays. Results: Anti-hypothalamic autoantibodies were found in AN patients. Furthermore, increased levels of oxidative stress were reported, as measured by decreased serum uric acid and total antioxidant capacity (TAC), and they reduced with the disease duration and the restoration of body mass index (BMI). Finally, a decrease in both autoantibodies and oxidative stress was observed as patients' clinical condition improved, as measured by time since diagnosis and BMI recovery. Conclusions: The clinical improvement of AN patients seems to be associated with a decrease in the autoimmune response to hypothalamic cellular antigens and a reduction in oxidative stress. Dysregulation of the immune system and oxidative stress appear to be interconnected in various diseases, including autoimmune and psychiatric disorders. These findings, although preliminary, may offer potential avenues for the treatment of this challenging condition.

Background. Naturally occurring human antibodies against glycans recognize and quickly eliminate infectious bacteria, viruses and aberrantly glycosylated neoplastic malignant cells, and they often initiate processes that involve the complement system. Methods. Using a printed glycan array (PGA) containing 605 glycoligands (oligo- and polysaccharides, glycopeptides), we examined which of the glycan-binding antibodies are able to activate the complement system. Using this PGA, the specificities of antibodies of the IgM and IgG classes were determined in the blood serum of healthy donors (suggested as mostly natural), and, then, using the same array, it was determined which types of the bound immunoglobulins were also showing C3 deposition. Results. It was found that about 30% of anti-glycan antibodies in human serum detected by the PGA did not activate the complement. They were mostly IgGs and directed to bacterial O-antigens; no apparent common structural motif within their target polysaccharides was found. Antibodies to blood group systems ABO and Forssman, xeno-antigens, a number of polysaccharides from various strains of S. enterica, E. coli and P. alcalifaciens, as well as small fragments of bacterial polysaccharides were recognized by complement-activating antibodies as expected. A complement-activating antibody was affinity-isolated on glycan-Sepharose from human serum, and, in the presence of the complement, it lysed red blood cells coated with the same glycan (kodecytes, where glycans expressed on biological membranes), while an isolated complement non-activating antibody did not, which confirms the validity of the solid-phase PGA results. Conclusions. Thus, ~30% of human anti-glycan antibodies lack the ability to activate the complement system. The function of the widely represented immunoglobulins that do not cause C3 deposition remains unclear.

Background: Rigorous assessment of antibody developability is crucial for optimizing lead candidates before progressing to clinical studies. Recent advances in predictive tools for protein structures, surface properties, stability, and immunogenicity have streamlined the development of new biologics. However, accurate prediction of the impact of single amino acid substitutions on antibody structures remains challenging, due to the diversity of complementarity-determining regions (CDRs), particularly CDR3s.

Methods: In this study, we combined in silico tools with in vitro assessments to engineer improved antibodies against the oxidized isoform of the macrophage migration inhibitory factor (oxMIF), building on the first generation anti-oxMIF antibody imalumab.

Results: We identified hydrophobic hotspots conferring increased self-interaction and aggregation propensity on imalumab, which unravels its unusually short half-life in humans. By introducing mutations into the variable regions, we addressed these liabilities. Structural prediction tools and molecular dynamics simulations guided the selection of mutations, which were then experimentally validated. The lead candidate antibody, C0083, demonstrated reduced hydrophobicity and self-interaction due to the restructuring of its heavy chain CDR3 loop. Despite these structural changes, C0083 retained target specificity and binding affinity to oxMIF.

Conclusions: Altogether, this study shows that a small number of well-selected mutations was sufficient to substantially improve the biophysicochemical properties of imalumab.

Background/objectives: Pemphigus comprises a diverse group of disorders within the autoimmune bullous dermatoses (AIBDs) spectrum. Among these, pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are the most commonly encountered variants. Despite its rarity, this condition can pose a life-threatening risk. We aimed to assess clinical characteristics, comorbidities, medication, as well as the treatment of various types of pemphigus in pemphigus patients.

Methods: We gathered data from 69 patients treated in the Department of Dermatology in the years 2016-2023. The investigation included sex, age at diagnosis, type of pemphigus, comorbidities and medications, presence of neoplasms and treatment of pemphigus, as well as enzyme-linked immunosorbent assay (ELISA) and direct immunofluorescence (DIF) results. The data were statistically analyzed with the p-value set at 0.05.

Results: The study group comprised 69 patients, including 41 women and 28 men. The mean age at diagnosis was 56.89 years +/- 15.42 years. A total of 79.31% of the patients were diagnosed with PV and the following 26.09% with PF. The most common comorbidities were arterial hypertension, hypercholesterolemia, and diabetes mellitus. The dominant treatment regimen was the systemic use of glucocorticosteroids (GCSs; 90% and 94% of PV and PF patients, respectively). More than half of the patients received at least one GCS-sparing treatment, including dapsone and rituximab. We observed a significantly frequent presence of IgG deposits in DIF in patients with PF (p = 0.0217) and a subsequent correlation between the concurrent presence of IgG deposits in DIF and anti-DSG1 antibodies in ELISA testing (p = 0.0469). The combination of IgG, IgG1, IgG4, and C3 deposits was more often existent in PF patients (p = 0.0054) and the combination of IgG4 and C3 deposits in PV patients (p = 0.0339). We also found a positive correlation between the level of anti-DSG1 antibodies and the age at diagnosis (p = 0.0298).

Conclusions: Patients with pemphigus are very often diagnosed with significant comorbidities and take diverse medication, which shows that the treatment of pemphigus should follow a multidisciplinary approach. Accurate analysis of the clinical condition of the patients, as well as the results of the ELISA panel or DIF, is crucial for a successful diagnostic and therapeutic process.