Characterization of structural and functional properties of anti-VEGF-IgG molecules under certain environmental stress conditions

Monoclonal antibodies (mAbs) are protein-structured molecules that bind to the target molecule with high specificity and have practical biotherapeutic effects. They are produced for therapeutic purposes in the pharmaceutical industry due to their essential functions, such as antigen-dependent cell cytotoxicity (ADCC), complement-dependent cell cytotoxicity (CDC), and target molecule neutralization. However, due to the protein nature of mAbs, they can be exposed to many different environmental stress factors during and after production. In the presence of these stress factors, the structures of antibodies and, accordingly, their biological functions can be negatively affected. The mAbs are exposed to stress factors such as thermal, changing pH conditions, and oxidative environment during production and post-production processes such as shipping and storage. In the presence of these stress factors, in addition to the development of processes such as aggregation and fragmentation in the structures of antibodies, they can undergo post-translational modifications (PTMs). These modifications and degradation processes can seriously affect the therapeutic activity of antibodies. Therefore, essential authorities in drug approval, such as the Food and Drug Administration (FDA), have made it mandatory to characterize the changes in the structure of mAbs and the changes in their biological activities in the presence of these stress factors. For this purpose, a large field such as forced degradation studies has been created. Based on these studies, the antibody's characterization and biological activities can be determined quickly by imitating the stress factors that mAbs may encounter under normal environmental conditions in the laboratory environment. Within the scope of this study, both structural and functional analyzes of the antibody were performed by forming the most common stress factors such as thermal, pH change of the environment, and oxidative stress of the anti-VEGF IgG molecule in vitro. Therefore, by characterizing the PTMs and aggregation profiles that can occur, many preliminary studies will be carried out for the therapeutic use of this antibody in the pharmaceutical industry, and it will lead to studies for many industrial and therapeutic purposes

A novel approach in cancer immunotherapy: natural killer cells expressing T cell receptors

T cell receptor (TCR) gene therapy is developed to redirect cytotoxic T cells towards selected epitopes of tumor antigens. However, due to the heterodimeric nature of the TCR molecule, alpha and beta chains introduced by gene delivery have a risk of pairing with the endogenously expressed complementary alpha or beta chains in the T cell. This phenomenon named as “mispairing” gives rise to TCRs of unpredictable specificity and causes potentially lethal side effects. Natural killer (NK) cells were discovered 40 years ago, by their ability to recognize and kill tumor cells without the requirement of prior immunization or stimulation. Since then, NK cells have grown to be promising agents for adoptive immunotherapy of cancer. In the last decade, several NK cell based anti-cancer products have been taken to clinical trials with promising results. However, to manufacture more efficient NK cell therapy products, it is essential to develop novel strategies to increase safety, efficiency and specificity with approaches such as retargeting NK cells against specific antigens which to date has only been possible with chimeric antigen receptors (CARs). In this thesis, we propose to use NK cells for TCR gene therapy aiming to reprogram them to selectively target tumor or virus antigens in complex with major histocompatibility complex. Our results convincingly demonstrate that the introduction of a functional TCR complex to NK cells via lentiviral gene transfer dramatically enhances the efficiency to mount antigen-specific cytotoxic activity. To our knowledge, the transfer of a TCR into an NK cell has never been reported before. Our strategy does not only have the potential to open up a whole new chapter in the field of cancer immunotherapy but also provides a final and definitive solution for the mispairing problem observed in TCR gene therapy

Analytical investigation of forced oxidized anti-VEGF IgG molecules: a focus on the alterations in antigen and receptor binding activities

Alterations in the biological activity of the molecules under stress conditions have not been documented as widely in the literature yet. This study was designed to reveal the functional impacts of various oxidation conditions on a model mAb, a commercial anti-VEGF IgG molecule. The responses to antigen binding, cell proliferation, FcRn receptors, and C1q binding, which rarely appear in the current literature, were investigated. The authors report peptide mapping data, post-translational modification (PTM) analysis, cell proliferation performance, and antigen (VEGF), C1q, and FcRn binding activities of the mAb under various stress conditions. The oxidation-prone site of the mAb was determined as Met252 in the DTLMISR peptide. The VEGF binding activity and anti-cell proliferation activity of the mAbs did not alter, while C1q and FcRn binding capacity significantly decreased under oxidative stress conditions. The full report is vital for many scientific and industrial processes about mAbs. The authors recommend performing functional analyses in addition to the structural studies while investigating the impacts of stress factors on therapeutic mAbs

SELEX against whole-cell bacteria resulted in lipopolysaccharide binding aptamers

Nucleic acid aptamers are target-specific oligonucleotides selected from combinatorial libraries through an iterative in vitro screening process known as Systemic Evolution of Ligands by Exponential Enrichment (SELEX). In this report, the selection of bacteria differentiating ssDNA aptamer candidates from a combinatorial library through the whole-cell SELEX method was performed. The enriched SELEX pool was sequenced using Illumina Next-Generation Sequencing (NGS) technology and analyzed for the most abundant sequences using CLC Genomics Workbench. The sequencing data resulted in several oligonucleotide families from which three individual sequences were chosen per SELEX based on the copy numbers. The binding performance of the selected aptamers was assessed by flow cytometry and fluorescence spectroscopy, and the binding constants were estimated using binding saturation curves. Varying results were obtained from two independent SELEX procedures where the SELEX against the model gram-negative bacterium Escherichia coli provided more selective sequences while the SELEX library used against gram-positive bacterium Listeria monocytogenes did not evolve as expected. The sequences that emerged from E. coli SELEX were shown to bind Lipopolysaccharide residues (LPS) and inhibit LPS-induced macrophage polarization. Thus, it can be said that, performed whole-cell SELEX could be resulted as the selection of aptamers which can bind LPS and inhibit LPS induced inflammation response and thus can be candidates for the inhibition of bacterial infections. In future studies, the selected aptamer sequences could be structurally and chemically modified and exploited as potential diagnostic tools and therapeutic agents as LPS antagonists

Influence of fluid properties on intensity of hydrodynamic cavitation and deactivation of salmonella typhimurium

In this study, three microfluidic devices with different geometries are fabricated on silicon and are bonded to glass to withstand high-pressure fluid flows in order to observe bacteria deactivation effects of micro cavitating flows. The general geometry of the devices was a micro orifice with macroscopic wall roughness elements. The width of the microchannel and geometry of the roughness elements were varied in the devices. First, the thermophysical property effect (with deionized water and phosphate-buffered saline (PBS)) on flow behavior was revealed. The results showed a better performance of the device in terms of cavitation generation and intensity with PBS due to its higher density, higher saturation vapor pressure, and lower surface tension in comparison with water. Moreover, the second and third microfluidic devices were tested with water and Salmonella typhimurium bacteria suspension in PBS. Accordingly, the presence of the bacteria intensified cavitating flows. As a result, both devices performed better in terms of the intensity of cavitating flow with the presence of bacteria. Finally, the deactivation performance was assessed. A decrease in the bacteria colonies on the agar plate was detected upon the tenth cycle of cavitating flows, while a complete deactivation was achieved after the fifteenth cycle. Thus, the proposed devices can be considered as reliable hydrodynamic cavitation reactors for "water treatment on chip" applications

Characterization of biological molecule–loaded nanostructures using circular dichroism and fourier transform infrared spectroscopy

Drug-loaded nanoparticles have many advantages in drug administration, which is an essential step for the impact of the drugs and their mechanism of action. Circular dichroism (CD) is a spectroscopy technique that measures the absorbance difference between right-circularly polarized light and left-circularly polarized light. Of several analytical techniques available, Fourier transform infrared spectroscopy is a powerful and widely employed technique explicitly for identifying chemical species. The peaks in the IR spectrum of a sample represent the molecular vibrations of the molecules present in the sample, signifying the various chemical bonds and functional groups. Various types of nanoparticles are being utilized for drug delivery applications because of their advantages such as controlled drug release, protection of the therapeutic payload, improved bioavailability, and targeted delivery. The discovery of novel nanoparticles and their application to the diagnosis and treatment of diseases have received considerable attention during the past decades

Optimized methods for analytical and functional comparison of biosimilar mAb drugs: a case study for Avastin, Mvasi, and Zirabev

Bevacizumab is a humanized therapeutic monoclonal antibody used to reduce angiogenesis, a hallmark of cancer, by binding to VEGF-A. Many pharmaceutical companies have developed biosimilars of Bevacizumab in the last decade. The official reports provided by the FDA and EMA summarize the analytical performance of biosimilars as compared to the originators without giving detailed analytical procedures. In the current study, several key methods were optimized and reported for analytical and functional comparison of bevacizumab originators (Avastin, Altuzan) and approved commercial biosimilars (Zirabev and Mvasi). This case study presents a comparative analysis of a set of biosimilars under optimized analytical conditions for the first time in the literature. The chemical structure of all products was analyzed at intact protein and peptide levels by high-resolution mass spectrometry; the major glycoforms and posttranslational modifications, including oxidation, deamidation, N-terminal PyroGlu addition, and C-terminal Lys clipping, were compared. The SPR technique was used to reveal antigen and some receptor binding kinetics of all products, and the ELISA technique was used for C1q binding affinity analysis. Finally, the inhibition performance of the samples was evaluated by an MTS-based proliferation assay in vitro. Major glycoforms were similar, with minor differences among the samples. Posttranslational modifications, except C-terminal Lys, were determined similarly, while unclipped Lys percentage was higher in Zirabev. The binding kinetics for VEGF, FcRn, FcγRIa, and C1q were similar or in the value range of originators. The anti-proliferative effect of Zirabev was slightly higher than the originators and Mvasi. The analysis of biosimilars under the same conditions could provide a new aspect to the literature in terms of the applied analytical techniques. Further studies in this field would be helpful to better understand the inter-comparability of the biosimilars

Fractionated charge variants of biosimilars: a review of separation methods, structural and functional analysis

The similarity between originator and biosimilar monoclonal antibody candidates are rigorously assessed based on primary, secondary, tertiary, quaternary structures, and biological functions. Minor differences in such parameters may alter target-binding, potency, efficacy, or half-life of the molecule. The charge heterogeneity analysis is a prerequisite for all biotherapeutics. Monoclonal antibodies are prone to enzymatic or non-enzymatic structural modifications during or after the production processes, leading to the formation of fragments or aggregates, various glycoforms, oxidized, deamidated, and other degraded residues, reduced Fab region binding activity or altered FcR binding activity. Therefore, the charge variant profiles of the monoclonal antibodies must be regularly and thoroughly evaluated. Comparative structural and functional analysis of physically separated or fractioned charged variants of monoclonal antibodies has gained significant attention in the last few years. The fraction-based charge variant analysis has proved very useful for the biosimilar candidates comprising of unexpected charge isoforms. In this report, the key methods for the physical separation of monoclonal antibody charge variants, structural and functional analyses by liquid chromatography-mass spectrometry, and surface plasmon resonance techniques were reviewed

Structural and Functional Analysis of CEX Fractions Collected from a Novel Avastin® Biosimilar Candidate and Its Innovator: A Comparative Study

Avastin® is a humanized recombinant monoclonal antibody used to treat cancer by targeting VEGF-A to inhibit angiogenesis. SIMAB054, an Avastin® biosimilar candidate developed in this study, showed a different charge variant profile than its innovator. Thus, it is fractionated into acidic, main, and basic isoforms and collected physically by Cation Exchange Chromatography (CEX) for a comprehensive structural and functional analysis. The innovator product, fractionated into the same species and collected by the same method, is used as a reference for comparative analysis. Ultra-Performance Liquid Chromatography (UPLC) ESI-QToF was used to analyze the modifications leading to charge heterogeneities at intact protein and peptide levels. The C-terminal lysine clipping and glycosylation profiles of the samples were monitored by intact mAb analysis. The post-translational modifications, including oxidation, deamidation, and N-terminal pyroglutamic acid formation, were determined by peptide mapping analysis in the selected signal peptides. The relative binding affinities of the fractionated charge isoforms against the antigen, VEGF-A, and the neonatal receptor, FcRn, were revealed by Surface Plasmon Resonance (SPR) studies. The results show that all CEX fractions from the innovator product and the SIMAB054 shared the same structural variants, albeit in different ratios. Common glycoforms and post-translational modifications were the same, but at different percentages for some samples. The dissimilarities were mostly originating from the presence of extra C-term Lysin residues, which are prone to enzymatic degradation in the body, and thus they were previously assessed as clinically irrelevant. Another critical finding was the presence of different glyco proteoforms in different charge species, such as increased galactosylation in the acidic and afucosylation in the basic species. SPR characterization of the isolated charge variants further confirmed that basic species found in the CEX analyses of the biosimilar candidate were also present in the innovator product, although at lower amounts. The charge variants’ in vitro antigen- and neonatal receptor-binding activities varied amongst the samples, which could be further investigated in vivo with a larger sample set to reveal the impact on the pharmacokinetics of drug candidates. Minor structural differences may explain antigen-binding differences in the isolated charge variants, which is a key parameter in a comparability exercise. Consequently, such a biosimilar candidate may not comply with high regulatory standards unless the binding differences observed are justified and demonstrated not to have any clinical impact