Safety Of Therapeutic Proteins Research Articles

A mechanistic marker-based screening tool to predict clinical immunogenicity of biologics

BackgroundThe efficacy and safety of therapeutic proteins are undermined by immunogenicity driven by anti-drug antibodies. Immunogenicity risk assessment is critically necessary during drug development, but current methods lack predictive power and mechanistic insight into antigen uptake and processing leading to immune response. A key mechanistic step in T-cell-dependent immune responses is the migration of mature dendritic cells to T-cell areas of lymphoid compartments, and this phenomenon is most pronounced in the immune response toward subcutaneously delivered proteins.MethodsThe migratory potential of monocyte-derived dendritic cells is proposed to be a mechanistic marker for immunogenicity screening. Following exposure to therapeutic protein in vitro, dendritic cells are analyzed for changes in activation markers (CD40 and IL-12) in combination with levels of the chemokine receptor CXCR4 to represent migratory potential. Then a transwell assay captures the intensity of dendritic cell migration in the presence of a gradient of therapeutic protein and chemokine ligands.ResultsHere, we show that an increased ability of the therapeutic protein to induce dendritic cell migration along a gradient of chemokine CCL21 and CXCL12 predicts higher immunogenic potential. Expression of the chemokine receptor CXCR4 on human monocyte-derived dendritic cells, in combination with activation markers CD40 and IL-12, strongly correlates with clinical anti-drug antibody incidence.ConclusionsMechanistic understanding of processes driving immunogenicity led to the development of a predictive tool for immunogenicity risk assessment of therapeutic proteins. These predictive markers could be adapted for immunogenicity screening of other biological modalities.

A superior extracellular matrix binding motif to enhance the regenerative activity and safety of therapeutic proteins

Among therapeutic proteins, cytokines and growth factors have great potential for regenerative medicine applications. However, these molecules have encountered limited clinical success due to low effectiveness and major safety concerns, highlighting the need to develop better approaches that increase efficacy and safety. Promising approaches leverage how the extracellular matrix (ECM) controls the activity of these molecules during tissue healing. Using a protein motif screening strategy, we discovered that amphiregulin possesses an exceptionally strong binding motif for ECM components. We used this motif to confer the pro-regenerative therapeutics platelet-derived growth factor-BB (PDGF-BB) and interleukin-1 receptor antagonist (IL-1Ra) a very high affinity to the ECM. In mouse models, the approach considerably extended tissue retention of the engineered therapeutics and reduced leakage in the circulation. Prolonged retention and minimal systemic diffusion of engineered PDGF-BB abolished the tumour growth-promoting adverse effect that was observed with wild-type PDGF-BB. Moreover, engineered PDGF-BB was substantially more effective at promoting diabetic wound healing and regeneration after volumetric muscle loss, compared to wild-type PDGF-BB. Finally, while local or systemic delivery of wild-type IL-1Ra showed minor effects, intramyocardial delivery of engineered IL-1Ra enhanced cardiac repair after myocardial infarction by limiting cardiomyocyte death and fibrosis. This engineering strategy highlights the key importance of exploiting interactions between ECM and therapeutic proteins for developing effective and safer regenerative therapies.

Correlative N-glycan and charge variant analysis of cetuximab expressed in murine, chinese hamster and human expression systems

A well-defined and controlled glycosylation pattern is important to maintain quality and safety of therapeutic proteins. Glycosylation is strongly dependent on the host cell line used for recombinant protein expression. Cetuximab, which is produced in mouse myeloma cells has been shown to harbour Fab glycans, which contain non-human like features and hence, can potentially cause an immunogenic response in patients. In light of the advent of biosimilar and biobetter development, we produced cetuximab variants in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. A combination of orthogonal chromatographic modes such as hydrophilic interaction, size exclusion and strong cation exchange chromatography with various detection strategies was employed to characterise the three different cetuximab variants and to compare the in-house produced HEK and CHO variants with the reference drug product. While Fc galactosylation and sialic acid content of the drug product and the HEK variant were highly similar, the CHO product showed lower galactosylation on Fc glycans and a comparatively low sialic acid content in the Fab region. The elevated high-mannose content of CHO cetuximab also suggests potential rapid clearence from circulation. The combination of multiple chromatographic separation modes has proven powerful for the characterisation of expression system dependent protein quality attributes such as N-glycosylation.

Abstract 1866: Pharmacokinetics and biodistribution of formatted Affimer® biotherapeutics targeting PD-L1

Abstract Programmed Cell Death-Ligand 1 (PD-L1) is part of the immune checkpoint system involved in preventing autoimmunity. PD-L1 is upregulated on tumors cells and binds to its receptor, PD-1, expressed by immune cells in the tumor microenvironment. Anti-PD-L1 immunotherapies have shown to be effective treatments both as monotherapies and in combinations with chemotherapies and radiotherapies, providing long term responses in a subset of patients. We have developed a PD-L1 Affimer antagonist that could differentiate itself from the current clinically approved mAbs due to its smaller size, improved tissue penetration and alternative routes of delivery such as a subcutaneous injection. It also opens up the possibility of generating additional PD-L1 antagonist therapeutics such as bispecifics and cytokine fusions. The Affimer biotherapeutic scaffold is a 14kDa monomeric protein based on the human protease inhibitor Stefin A, lacking post-translational modifications such as disulfide bonds and glycosylation. We have identified a range of high-affinity competitive binders to human PD-L1 confirmed by SPR using Fc formatted antigen, competition ELISA and blockade of PD-L1/PD-1 axis using various cell-based assays. We demonstrated efficacy in a humanized PD-L1 MC38 tumor growth inhibition model and compared it to approved PD-L1 antibody therapies. Pharmacokinetics (PK) and biodistribution are key parameters that influence the efficacy and safety of therapeutic proteins. Half-life extension of lead Affimer proteins was achieved by fusing to a human Fc region of an IgG (designated AVA004).Pharmacokinetic analysis of AVA004 Fc molecules were performed in C57Bl/6, humanized FcRn/HSA mice and cynomolgus after single intravenous administration. Tissue distribution following intravenous administration of AVA004 Fc to humanized NOG mice bearing orthotopic MDA-MB-231 tumor cells was evaluated using radiolabeled 125I- AVA004 Fc. Biodistribution imaging studies were conducted in mice bearing A375 tumor cells using IRDye800 conjugated to AVA004 Fc followed by ex vivo imaging. A more quantitative biodistribution study was conducted in the same model using SPECT imaging DOTAGA bioconjugated AVA004 Fc molecules radiolabeled with 111Indium. The imaging biodistribution allowed us to select the most favorable molecule based on its tumor/plasma and tumor/liver ratios. Citation Format: Estelle Adam, Emma Jenkins, Floriane Laurent, Jennifer Hillman, Maureen West, Emma Stanley, David Sanford, Agnieszka Kownacka, Céline Mothes, Alexandra Oudot, Vincent Matthew, David Gilfoyle, Barry Jones, William Bachovchin, Amrik Basran, Cyril Berthet. Pharmacokinetics and biodistribution of formatted Affimer® biotherapeutics targeting PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1866.

Generation of site-distinct N-glycan variants for in vitro bioactivity testing.

Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

Introduction: Proceedings of the 2015 Viral Clearance Symposium.

Viral clearance and inactivation by the bulk protein purification process are critical components of the overall strategy to ensure the safety of therapeutic proteins derived from mammalian cell culture. A mechanistic understanding of the factors that influence viral clearance and inactivation are

Unraveling the Effect of Immunogenicity on the PK/PD, Efficacy, and Safety of Therapeutic Proteins.

Biologics have emerged as a powerful and diverse class of molecular and cell-based therapies that are capable of replacing enzymes, editing genomes, targeting tumors, and more. As this complex array of tools arises a distinct set of challenges is rarely encountered in the development of small molecule therapies. Biotherapeutics tend to be big, bulky, polar molecules comprised of protein and/or nucleic acids. Compared to their small molecule counterparts, they are fragile, labile, and heterogeneous. Their biodistribution is often limited by hydrophobic barriers which often restrict their administration to either intravenous or subcutaneous entry routes. Additionally, their potential for immunogenicity has proven to be a challenge to developing safe and reliably efficacious drugs. Our discussion will emphasize immunogenicity in the context of therapeutic proteins, a well-known class of biologics. We set out to describe what is known and unknown about the mechanisms underlying the interplay between antigenicity and immune response and their effect on the safety, efficacy, pharmacokinetics, and pharmacodynamics of these therapeutic agents.

Measurement of Free Versus Total Therapeutic Monoclonal Antibody in Pharmacokinetic Assessment is Modulated by Affinity, Incubation Time, and Bioanalytical Platform.

Decisions about efficacy and safety of therapeutic proteins (TP) designed to target soluble ligands are made in part by their ex vivo quantification. Ligand binding assays (LBAs) are critical tools in measuring serum TP levels in pharmacokinetic, toxicokinetic, and pharmacodynamic studies. This study evaluated the impact of reagent antibody affinities, assay incubation times, and analytical platform on free or total TP quantitation. An ELISA-based LBA that measures monoclonal anti-sclerostin antibody (TPx) was used as the model system. To determine whether the method measures free or total TPx, the effects of K on, K off, and K D were determined. An 8:1 molar ratio of sclerostin (Scl) to TPx compared to a 1:1 molar ratio produced by rabbit polyclonal antibodies to TPx was required to achieve IC50, a measure of TPx interference effectiveness, making it unclear whether the ELISA truly measured free TPx. Kinetic analysis revealed that Scl had a rapid dissociation rate (K off) from TPx and that capture and detection antibodies had significantly higher binding affinities (K D) to TPx. These kinetic limitations along with long ELISA incubation times lead to the higher molar ratios (8:1) required for achieving 50% inhibition of TPx. However, a microfluidic platform with the same reagent pairs required shorter incubations to achieve a lower Scl IC50 molar ratio (1:1). The findings from this study provide the bioanalytical community with a deeper understanding of how reagent and platform selection for LBAs can affect what a particular method measures, either free or total TP concentrations.

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins

N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.

Mass spectrometric analysis of protein species of biologics

The global market for therapeutic proteins has been growing fast in the past decade. In comparison with conventional small-molecule drugs therapeutic proteins have a very complex chemical composition. These proteins are typically composed of dozens to hundreds of amino acids which are often post-translationally modified. Recombinant therapeutic proteins are produced by living organisms resulting in a heterogeneous mixture of recombinant protein species (isoforms, proteoforms). The pharmaceutical function and biological and toxicological properties of biologics and follow-on biosimilars are critically related to their exact chemical composition. Therefore, analytical tools capable of identifying and quantifying therapeutic protein species and their accompanying species, often differing in a few chemical moieties only, are necessary to guarantee efficacy and safety of therapeutic proteins. In this review we focus on the application of mass spectrometry for analyzing the exact chemical composition of therape...