Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a worldwide health problem. In a previous study, a murine monoclonal antibody (mMAB), capable of binding to PBP2a within MRSA strains, was generated. F(ab') antibody fragments are widely described in the literature as immunochemical tools and reagents for diagnostics and therapeutics, particularly because of their low immunogenicity and rapid pharmacokinetics. In this study, F(ab') fragments from mMAB were generated by enzymatic digestion, using pepsin. They were purified by affinity chromatography using protein A and concentrated by a MWCO 50 kDa filtration unit. The results indicate that it is possible to obtain F(ab') fragments by pepsin digestion. ELISA, western blotting, and fluorescence microscopy data demonstrated that F(ab') affinity for PBP2a is not lost even after the enzymatic digestion process. As expected, in the pharmacokinetics tests, F(ab') presented a faster elimination (between 12 and 18 h) compared to IgG. These F(ab') fragments could be used in future immunodiagnostic applications, including in vitro or in situ radiolabeling and in the treatment of infections caused by this important pathogen.
Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium responsible for serious nosocomial and community-acquired infections worldwide. Since few antibiotics are effective for treating MRSA infections, the development of new therapies is of great importance. Previous studies demonstrated that PBP2a is a target that generates protective antibodies against MRSA. A murine monoclonal antibody (MAb) that recognizes PBP2a from MRSA strains was previously isolated and characterized. In this report, we evaluated the biodistribution of this MAb in blood and tissues, as well as the extent of protection conferred using prophylactic and therapeutic assays compared to vancomycin treatment. Biodistribution was evaluated 12–96 h after MAb administration. It predominantly remained in the serum, but it was also detectable in the kidneys, lungs, and spleen at low concentrations (about 4.5% in the kidneys, 1.9% in the lungs, and 0.7% the spleen) at all observed timepoints. Prophylactic studies in a murine model demonstrated a significant bacterial load reduction in the kidneys of the groups treated with either with IgG (greater than 3 logs) or F(ab’)2 (98%) when compared to that of the control groups (untreated). Mice were challenged with a lethal dose, and the survival rate was higher in the treated mice. Treatment with the MAb resulted in a bacterial load reduction in the kidneys similar to that of mice treated with vancomycin, and a MAb/vancomycin combination therapy was also effective. These results demonstrate that an anti-PBP2a MAb may be a promising therapeutic for treating MRSA infections.
Introduction: Acinetobacter baumannii is an important opportunistic pathogen, with high incidence in intensive care units, mainly affecting immunocompromised patients. The increasing resistance to β-lactam antibiotics, especially to carbapenems, complicates the treatment and raises the need for novel therapy approaches, such as immunotherapies. Development of monoclonal antibodies (mAbs) against multi-drug resistant bacteria is well established in the literature. However, their true potential has not been fully tapped. In general, mAbs present high pathogen specificity, favorable pharmacokinetics with great versatility, allowing their use to not only immunotherapies but also in immunodiagnostics.Objective: Regarding the multi-drug resistance problematic, this study aims to develop an anti-A. baumannii monoclonal antibody.Methodology: First, three female BALB/c mice were immunized intraperitoneally with 25µg of a recombinant A. baumannii protein and Freund's adjuvant every 2 weeks. Mouse splenocytes were then isolated after the fourth immunization, fused with SP2/0 murine myeloma cells to obtain hybridomas and ELISA was used for screening. Briefly, 5 µg/mL of recombinant protein in 50 mM carbonate-bicarbonate buffer was used as the coating agent, followed by incubation with hybridoma supernatants. Preimmune mice sera were used as negative controls, and binding of antibodies to the recombinant protein was detected by goat anti-mouse IgG-peroxidase conjugates. TMB was used as substrate and reading was performed with absorbance at 450nm. Hybridomas that produced positively bound antibodies were cloned with limiting dilution and grown in DMEM FBS 20%/HT medium. Isotypes of the mAbs were determined using Pierce® rapid isotyping kits and western blot assays demonstrated mAb recognition to A. baumannii proteins.Results: Only one of the three animals generated stable antibody-producing hybridomas. From these cells, the ones with ELISA absorbance values higher than 2.0 were selected for cloning. Hence, 9 polyclonal hybridomas were cloned, where six were recloned in a way to guarantee their stability. All presented themselves as IgG1 subtype, except one (NG4) that had IgG2a isotype. In the western blot assays, it was observed antibody recognition for the recombinant protein in all, except one, of the tested supernatants. In contrast, eight supernatants were tested against A. baumannii lysate proteins. From these, two showed no recognition, while four presented a discrete binding to one protein in the bacterial lysate, and two demonstrated excellent antigenic recognition to a single protein of A. baumannii. Conclusion:Data together demonstrate that it was possible to obtain antibody-producing hibridomas that recognizes the target protein of A. baumannii, whether in the recombinant or the native form. These antibodies may have a potential use in therapy or diagnostics, but it all depends on how the antibody interacts with its target. Therefore, more screening and characterization tests are necessary until the complete establi...
Introduction: Biosimilars are complex biological products with a high degree of similarity in terms of the structural, functional, biological, and clinical attributes, to reflect the safety and efficacy of reference products (RP). Biosimilars could lead to more affordable treatments and increase patient access to expensive therapies. In this sense, Quality by design (QbD) plays an important role as a modern approach based on scientific data and risk management aiming to attend the quality pattern of an end-product. For biosimilars, QbD assumes an information-driven process that uses all available knowledge on the RP, mitigating potential risks and guiding to a more assertive process development.Objective: Develop and validate the use of the QbD approach in biosimilar development projects, applying this method in the development process of the biosimilar monoclonal antibodies (mAbs) nivolumab and pembrolizumab.Methodology: QbD process began with the determination of quality target product profile (QTTP) and critical quality attributes (CQAs). The establishment of these parameters were defined using information of the RP Keytruda and Opdivo and other registered mAbs. After the QTTP definition, the potential CQAs were assessed using a risk ranking and a filtering (RRF) approach developed by Roche/Genentech®. The RRF method evaluated each attribute according to impact score based on bioactivity, pharmacokinetics/ pharmacodynamics, immunogenicity or safety, and the uncertainty of the impact, to identify final CQAs.Results: QTTP for the biosimilars were assessed based on RP characteristics, considering their own production process. The CQAs were divided in structure, physicochemical and biological activity. Orthogonal methods were determined to measure similarity between biosimilar and RP, according to the Bio-Manguinhos reality. As described for other mAbs, some CQAs were obligatory due to regulatory requirements, as sterility and API concentration. As expected, the absence of detailed information in clinical trials studies increased the uncertainty score, leading to a high score impact of most CQAs. Structural parameters that have a higher impact in product quality provided us more information to a better assessment of the risks, leading to smaller scores. A final multidisciplinary brainstorm meeting shall validate these results. Conclusion:QbD is an important approach to guide biosimilars development at Bio-Manguinhos, as it helped to a strategic preparation at the very beginning of the projects. More information will be acquired along the biosimilars' development to assure a continuous evaluation and risk prioritization of CQAs.
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