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The present study was carried out to develop cisplatin-loaded chitosan nanoparticles (CCNP) and cisplatin-loaded chitosan nanoparticle surface linked to rituximab (mAbCCNP) as targeted delivery formulations. The two formulations (CCNP and mAbCCNP) exhibited significant physicochemical properties. The zetapotential (ZP) values of CCNP and mAbCCNP were 30.50 ± 5.64 and 26.90 ± 9.09 mV, respectively; while their particle sizes were 308.10 ± 1.10 and 349.40 ± 3.20 z.d.nm, respectively. The poly dispersity index (PDI) of CCNP was 0.257 ± 0.030 (66.6% PDI), while that of mAbCCNP was 0.444 ± 0.007 (57.60% PDI). Differential scanning calorimetry (DSC) revealed that CCNP had endothermic peaks at temperatures ranging from 135.50 to 157.69 °C. A sharp exothermic peak was observed at 95.79 °C, and an endothermic peak was observed at 166.60 °C. The XRD study on CCNP and mAbCCNP revealed distinct peaks at 2θ . Four peaks at 35.38°, 37.47°, 49.29°, and 59.94° corresponded to CCNP, while three distinct peaks at 36.6°, 49.12°, and 55.08° corresponded to mAbCCNP. The in vitro release of cisplatin from nanoparticles followed zero order kinetics in both CCNP and mAbCCNP. The profile for CCNP showed 43.80% release of cisplatin in 6 h (R 2  = 0.9322), indicating linearity of release with minimal deviation. However, the release profile of mAbCCNP showed 22.52% release in 4 h (R 2  = 0.9416), indicating linearity with sustained release. In vitro cytotoxicity studies on MCF-7 ATCC human breast cancer cell line showed that CCNP exerted good cytotoxicity, with IC 50 of 4.085 ± 0.065 µg/mL. However, mAbCCNP did not elicit any cytotoxic effect. At a dose of 4.00 µg/mL cisplatin induced early apoptosis and late apoptosis, chromatin condensation, while it produced secondary necrosis at a dose of 8.00 µg/mL. Potential delivery system for cisplatin CCNP and mAbCCNP were successfully formulated. The results indicated that CCNP was a more successful formulation than mAbCCNP due to lack of specificity of rituximab against MCF-7 ATCC human breast cancer cells.

Monoclonal antibodies (mAb) are key therapeutic agents in cancer immunotherapy and exert their effects through Fc receptor-dependent and -independent mechanisms. However, the nanoscale receptor reorganization resulting from mAb binding and its implications for the therapeutic mode of action remain poorly understood. Here, we present a multi-target 3D RESI super-resolution microscopy technique that directly visualizes the structural organization of CD20 receptors and the Type I (e.g., Rituximab) and Type II (e.g., Obinutuzumab) anti-CD20 therapeutic antibodies and quantitatively analyze these interactions at single-protein resolution in situ. We discover that, while Type I mAbs promote higher-order CD20 oligomerization, Type II mAbs induce limited clustering, leading to differences in therapeutic function. Correlating RESI with functional studies for Type II antibodies with different hinge region flexibilities, we show that the oligomeric CD20 arrangement determines the Type I or Type II function. Thus, the nanoscale characterization of CD20-mAb complexes enhances our understanding of the structure-function relationships of therapeutic antibodies and offers insights into the design of next-generation mAb therapies. The nanoscale organization of the antigen-antibody complexes influences the therapeutic action of monoclonal antibodies. Here, the authors present a multi-target 3D RESI imaging assay for the nanometer spatial analysis of CD20 in complex with therapeutic monoclonal antibodies within intact cells, to analyse the interdependency between the mode of antibody binding and the therapeutic function.

Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.

IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.

PurposePolysorbate excipients are commonly used as surfactants to stabilize therapeutic proteins in formulations. Degradation of polysorbates could lead to particle formation and instability of the drug formulation. We investigated how the fatty acid composition of polysorbate 80 impacts the degradation profile, particle formation, and product stability under stress conditions.MethodsTwo polysorbate 80-containing therapeutic protein formulations were reformulated with either Polysorbate 80 NF synthesized from a fatty acid mixture that contains mainly oleic acid (≥58%) or a version of polysorbate 80 synthesized with high oleic acid (>98%). Stress conditions, including high temperature and esterase spiking, were applied and changes to both the polysorbate and the therapeutic protein product were investigated for stability, purity, innate immune response and biological activity.ResultsThe addition of esterase and storage at 37°C led to significant hydrolysis of the polysorbate and increases in sub-visible particle formation for both polysorbates tested. The fatty acid composition of polysorbate 80 did not directly alter the stability profile of either therapeutic protein as measured by size exclusion chromatography, or significantly impact innate immune response or biological activity. However, formulations with Polysorbate 80 NF showed greater propensity for sub-visible particle formation under stress conditions.ConclusionsThese results suggest that composition of fatty acids in polysorbate 80 may be a promoter for sub-visible particulate formation under the stress conditions tested but may not impact protein aggregation or biological activity.

Visible particulate (VP) matter in therapeutic protein drug products (DPs) is a quality attribute that can impact product efficacy and patient safety across the product lifecycle. This study evaluates Morphologically Directed Raman Spectroscopy (MDRS) for detecting and characterizing VPs in therapeutic protein DPs. MDRS combines morphological and chemical analyses, which are essential for root cause analysis (RCA) during incident investigations of VP formation. We evaluated MDRS using polystyrene (PS) microsphere standards, candidate reference materials from the US National Institute of Standards and Technology (NIST), and stressed therapeutic protein DPs, insulin lispro and rituximab. Using MDRS, VPs were imaged and chemically characterized in insulin or rituximab drug products with varying morphologies and ranged from 100 to 400 μm in diameter. The intensity of Raman spectral peaks varied with VP size, transparency, morphological complexity, and between therapeutic protein drug classes. Principal component analysis (PCA) of morphological features (e.g., size, shape, and transparency) revealed that NIST ethylene tetrafluoroethylene (ETFE) particles displayed a range of morphologies, akin to stressed therapeutic protein VPs. These results highlight the potential of combining morphological analysis with Raman spectroscopy to support RCA in therapeutic protein DPs for both pre- and post-market quality assessments.

Complete characterization and quantification of monoclonal antibodies often rely on enzymatic digestion with trypsin. In order to accelerate and automate this frequently performed sample preparation step, immobilized enzyme reactors (IMER) compatible with standard HPLC systems were used. This allows an automated online approach in all analytical laboratories. We were able to demonstrate that the required digestion time for the model monoclonal antibody rituximab could be reduced to 20 min. Nevertheless, a previous denaturation of the protein is required, which also needs 20 min. Recoveries were determined at various concentrations and were 100% ± 1% at 100 ng on column, 96% ± 7% at 250 ng on column and 98% ± 2% at 450 ng on column. Despite these good recoveries, complete digestion was not achieved, resulting in a poorer limit of quantification. This is 50 ng on column under optimized IMER conditions, whereas an offline digest on the same system achieved 0.3 ng on column. Furthermore, our work revealed that TRIS buffers, when used with an IMER system, led to alteration of the peptides and induced modifications in the peptides. Therefore, the addition of TRIS should be avoided when working at elevated temperatures of about 60 °C. Nevertheless, our results have shown that the recovery is not significantly influenced whether TRIS is used or not (recovery: 96 ± 7% with TRIS vs. 100 ± 9% without TRIS).

Background One of the main problems in B cell lymphoma treatment is severe adverse effects and low therapeutic efficacy resulting from systemic chemotherapy. A pH-sensitive controlled drug release system based on mesoporous silica nanoparticles was constructed for targeted drug delivery to tumor cells to reduce systemic toxicity and improve the therapeutic efficacy. Methods In this study, the doxorubicin (DOX) was filled into the mesopores of the functional MSNs (DMSNs). Furthermore, rituximab was introduced as the targeted motif of functional DMSNs using an avidin-biotin bridging method to evaluate the targetability to tumor cells. Then, the cell viability and apoptosis efficiency after treatment with rituximab-conjugated DMSNs (RDMSNs) were estimated by using CCK-8 assay and flow cytometry, respectively. Additionally, the research in vivo was performed to evaluate the enhanced antitumor efficacy and the minimal toxic side effects of RDMSNs. Also, TUNEL staining assay was employed to explore the mechanism of antitumor effects of RDMSNs. Results This targeted drug delivery system exhibited low premature drug release at a physiological pH and efficient pH-responsive intracellular release under weakly acidic conditions. The in vitro tests confirmed that targeted RDMSNs could selectively adhere to the surface of lymphoma B cells via specific binding with the CD20 antigen and be internalized into CD20 positive Raji cells but few CD20 negative Jurkat cells, which leads to increased cytotoxicity and apoptosis of the DOX in Raji cells due to the release of the entrapped DOX with high efficiency in the slightly acidic intracellular microenvironment. Furthermore, the in vivo investigations confirmed that RDMSNs could efficiently deliver DOX to lymphoma B cells by pH stimuli, thus inducing cell apoptosis and inhibiting tumor growth, while with minimal toxic side effects. Conclusions This targeted and pH-sensitive controlled drug delivery system has the potential for promising application to enhance the therapeutic index and reduce the side effects of B cell lymphoma therapy.

Autoimmune endocrine disorders, such as type 1 diabetes (T1D) and thyroiditis, at present are treated with only hormone replacement therapy. This emphasizes the need to identify personalized effective immunotherapeutic strategies targeting T and B lymphocytes. Among the genetic variants associated with several autoimmune disorders, the C1858T polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene, encoding for Lyp variant R620W, affects the innate and adaptive immunity. We previously exploited a novel personalized immunotherapeutic approach based on siRNA delivered by liposomes (lipoplexes) that selectively inhibit variant allele expression. In this manuscript, we improved lipoplexes carrying siRNA for variant C1858T by functionalizing them with Fab of Rituximab antibody (RituxFab-Lipoplex) to specifically target B lymphocytes in autoimmune conditions, such as T1D. RituxFab-Lipoplexes specifically bind to B lymphocytes of the human Raji cell line and of human PBMC of healthy donors. RituxFab-Lipoplexes have impact on the function of B lymphocytes of T1D patients upon CpG stimulation showing a higher inhibitory effect on total cell proliferation and IgM+ plasma cell differentiation than the not functionalized ones. These results might open new pathways of applicability of RituxFab-Lipoplexes, such as personalized immunotherapy, to other autoimmune disorders, where B lymphocytes are the prevalent pathogenic immunocytes.

Biosimilars are highly similar to, but not identical with, their originator products. As a result, structural differences between originators and biosimilars can be difficult to detect and characterize without the appropriate analytical tools. Therefore, we first focus on identifying initial structural differences between rituximab, bevacizumab, and trastuzumab originator and biosimilar pairs and later address how these differences change after applying thermal stress at 40 °C with orbital shaking for 4 weeks. Prior to incubation, we detected comparable secondary and tertiary structures for each pair and identified different levels of soluble aggregates, charge variants, and molecular weight variants due to differences in glycoforms and the number of C-terminal lysine groups. Over the course of incubation, we compared differences in charge variants and unfolding patterns. Taken together, our study provides a comparability exercise, providing information on the minor differences present between originator and biosimilar products and how those differences are impacted by stress.