Trace Levels of Innate Immune Response Modulating Impurities (IIRMIs) Synergize to Break Tolerance to Therapeutic Proteins

Therapeutic proteins such as monoclonal antibodies, replacement enzymes and toxins have significantly improved the therapeutic options for multiple diseases, including cancer and inflammatory diseases as well as enzyme deficiencies and inborn errors of metabolism. However, immune responses to these products are frequent and can seriously impact their safety and efficacy. Of the many factors that can impact protein immunogenicity, this study focuses on the role of innate immune response modulating impurities (IIRMIs) that could be present despite product purification and whether these impurities can synergize to facilitate an immunogenic response to therapeutic proteins. Using lipopolysaccharide (LPS) and CpG ODN as IIRMIs we showed that trace levels of these impurities synergized to induce IgM, IFNγ, TNFα and IL-6 expression. In vivo, trace levels of these impurities synergized to increase antigen-specific IgG antibodies to ovalbumin. Further, whereas mice treated with human erythropoietin showed a transient increase in hematocrit, those that received human erythropoietin containing low levels of IIRMIs had reduced response to erythropoietin after the 1st dose and developed long-lasting anemia following subsequent doses. This suggests that the presence of IIRMIs facilitated a breach in tolerance to the endogenous mouse erythropoietin. Overall, these studies indicate that the risk of enhancing immunogenicity should be considered when establishing acceptance limits of IIRMIs for therapeutic proteins

Fluorescence Single Particle Tracking for the Characterization of Submicron Protein Aggregates in Biological Fluids and Complex Formulations

To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA). A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 mu m (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA. The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins. fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations

A short history of the 5-HT2C receptor: from the choroid plexus to depression, obesity and addiction treatment

This paper is a personal account on the discovery and characterization of the 5-HT2C receptor (first known as the 5- HT1C receptor) over 30 years ago and how it translated into a number of unsuspected features for a G protein-coupled receptor (GPCR) and a diversity of clinical applications. The 5-HT2C receptor is one of the most intriguing members of the GPCR superfamily. Initially referred to as 5-HT1CR, the 5-HT2CR was discovered while studying the pharmacological features and the distribution of [3H]mesulergine-labelled sites, primarily in the brain using radioligand binding and slice autoradiography. Mesulergine (SDZ CU-085), was, at the time, best defined as a ligand with serotonergic and dopaminergic properties. Autoradiographic studies showed remarkably strong [3H]mesulergine-labelling to the rat choroid plexus. [3H]mesulergine-labelled sites had pharmacological properties different from, at the time, known or purported 5-HT receptors. In spite of similarities with 5-HT2 binding, the new binding site was called 5-HT1C because of its very high affinity for 5-HT itself. Within the following 10 years, the 5-HT1CR (later named 5- HT2C) was extensively characterised pharmacologically, anatomically and functionally: it was one of the first 5-HT receptors to be sequenced and cloned. The 5-HT2CR is a GPCR, with a very complex gene structure. It constitutes a rarity in theGPCR family: many 5-HT2CR variants exist, especially in humans, due to RNA editing, in addition to a few 5-HT2CR splice variants. Intense research led to therapeutically active 5-HT2C receptor ligands, both antagonists (or inverse agonists) and agonists: keeping in mind that a number of antidepressants and antipsychotics are 5- HT2CR antagonists/inverse agonists. Agomelatine, a 5-HT2CR antagonist is registered for the treatment of major depression. The agonist Lorcaserin is registered for the treatment of aspects of obesity and has further potential in addiction, especially nicotine/ smoking. There is good evidence that the 5-HT2CR is involved in spinal cord injury-induced spasms of the lower limbs, which can be treated with 5-HT2CR antagonists/inverse agonists such as cyproheptadine or SB206553. The 5-HT2CR may play a role in schizophrenia and epilepsy. Vabicaserin, a 5-HT2CR agonist has been in development for the treatment of schizophrenia and obesity, but was stopped. As is common, there is potential for further indications for 5-HT2CR ligands, as suggested by a number of preclinical and/or genome-wide association studies (GWAS) on depression, suicide, sexual dysfunction, addictions and obesity. The 5-HT2CR is clearly affected by a number of established antidepressants/antipsychotics and may be one of the culprits in antipsychotic-induced weight gain

Integration of Motion Responses Underlying Directional Motion Anisotropy in Human Early Visual Cortical Areas

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