New Opportunities in Glycan Engineering for Therapeutic Proteins

Glycans as sugar polymers are important metabolic, structural, and physiological regulators for cellular and biological functions. They are often classified as critical quality attributes to antibodies and recombinant fusion proteins, given their impacts on the efficacy and safety of biologics drugs. Recent reports on the conjugates of N-acetyl-galactosamine and mannose-6-phosphate for lysosomal degradation, Fab glycans for antibody diversification, as well as sialylation therapeutic modulations and O-linked applications, have been fueling the continued interest in glycoengineering. The current advancements of the human glycome and the development of a comprehensive network in glycosylation pathways have presented new opportunities in designing next-generation therapeutic proteins.

TCDD Inhibition of IgG1 Production in Experimental Autoimmune Encephalomyelitis (EAE) and In Vitro

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) is a ligand for the aryl hydrocarbon receptor (AhR). TCDD is well-characterized to produce immunotoxicity, including suppression of antibody production. Previously we showed that TCDD inhibited myelin oligodendrocyte glycoprotein (MOG) peptide-specific IgG and attenuated disease in experimental autoimmune encephalomyelitis (EAE) model in mice. Thus, the purpose of this study was to characterize the effects of TCDD on IgG subclasses in EAE and in vitro and assess effects in B cells derived from various tissues. TCDD modestly suppressed intracellular IgG expression in splenocytes (SPLC), but not bone marrow (BM) or lymph node (LN) cells. To further understand TCDD’s effects on IgG, we utilized LPS and LPS + IL-4 in vitro to stimulate IgG3 and IgG1 production, respectively. TCDD preferentially suppressed IgG1+ cell surface expression, especially in SPLC. However, TCDD was able to suppress IgG1 and IgG3 secretion from SPLC and B cells, but not BM cells. Lastly, we revisited the EAE model and determined that TCDD suppressed MOG-specific IgG1 production. Together these data show that the IgG1 subclass of IgG is a sensitive target of suppression by TCDD. Part of the pathophysiology of EAE involves production of pathogenic antibodies that can recruit cytolytic cells to destroy MOG-expressing cells that comprise myelin, so inhibition of IgG1 likely contributes to TCDD’s EAE disease attenuation.

Immune Maturation Effects on Viral Neutralization and Avidity of Hyperimmunized Equine Anti-SARS-CoV-2 Sera

Mass-vaccination against COVID-19 is still a distant goal for most low-to-middle income countries. The experience gained through decades producing polyclonal immunotherapeutics (such as antivenoms) in many of those countries is being redirected to develop similar products able to neutralize SARS-CoV-2 infection. In this study we analyzed the biological activity (viral neutralization or NtAb) and immunochemical properties of hyperimmune horses’ sera (HHS) obtained during initial immunization (I) and posterior re-immunization (R) cycles using the RBD domain of the SARS-CoV-2 spike protein as antigen. HHS at the end of the R cycle showed higher NtAb titers when compared to those after the I cycle (35,585 vs. 7000 mean NtAb, respectively). Moreover, this increase paralleled an increase in avidity (95.2% to 65.2% mean avidity units, respectively). The results presented herein are relevant for manufacturers of these therapeutic tools against COVID-19.

Pharmacokinetic Developability and Disposition Profiles of Bispecific Antibodies: A Case Study with Two Molecules

Bispecific antibodies (BsAb) that engage multiple pathways are a promising therapeutic strategy to improve and prolong the efficacy of biologics in complex diseases. In the early stages of discovery, BsAbs often exhibit a broad range of pharmacokinetic (PK) behavior. Optimization of the neonatal Fc receptor (FcRn) interactions and removal of undesirable physiochemical properties have been used to improve the ‘pharmacokinetic developability’ for various monoclonal antibody (mAb) therapeutics, yet there is a sparsity of such information for BsAbs. The present work evaluated the influence of FcRn interactions and inherent physiochemical properties on the PK of two related single chain variable fragment (scFv)-based BsAbs. Despite their close relation, the two BsAbs exhibit disparate PK in cynomolgus monkeys with BsAb-1 having an aberrant clearance of ~2 mL/h/kg and BsAb-2 displaying a an ~10-fold slower clearance (~0.2 mL/h/kg). Evaluation of the physiochemical characteristics of the molecules, including charge, non-specific binding, thermal stability, and hydrophobic properties, as well as FcRn interactions showed some differences. In-depth drug disposition results revealed that a substantial disparity in the complete release from FcRn at a neutral pH is a primary factor contributing to the rapid clearance of the BsAb-1 while other biophysical characteristics were largely comparable between molecules.

Impact of Intrinsic and Extrinsic Factors on the Pharmacokinetics of Peptides: When Is the Assessment of Cer-Tain Factors Warranted?

Peptides are short chains of 2 to 50 amino acids (molecular weight of less than 10 kDa) linked together by peptide bonds. As therapeutic agents, peptides are of interest because the body naturally produces many different peptides. Short-chain peptides have many advantages as compared with long-chain peptides (e.g., low toxicity). The first peptide corticotropin was approved in 1952 for multiple inflammatory diseases and West syndrome. Since then, more than 60 peptides have been approved by the FDA. Pharmacokinetics (PK) is widely used in modern-day drug development for designing a safe and efficacious dose to treat a wide variety of diseases. There are, however, several factors termed as “intrinsic” or “extrinsic” which can influence the PK of a drug, and as a result, one has to adjust the dose in a patient population. These intrinsic and extrinsic factors can be described as age, gender, disease states such as renal and hepatic impairment, drug–drug interaction, food, smoking, and alcohol consumption. It is well known that these intrinsic and extrinsic factors can have a substantial impact on the PK of small molecules, but for macromolecules, the impact of these factors is not well established. This review summarizes the impact of intrinsic and extrinsic factors on the PK of peptides.

Disease Prevalence Matters: Challenge for SARS-CoV-2 Testing

While sensitivity and specificity are important characteristics for any diagnostic test, the influence of prevalence is equally, if not more, important when such tests are used in community screening. We review the concepts of positive/negative predictive values (PPV/NPV) and how disease prevalence affects false positive/negative rates. In low-prevalence situations, the PPV decreases drastically. We demonstrate how using two tests in an orthogonal fashion can be especially beneficial in low-prevalence settings and greatly improve the PPV of the diagnostic test results.

Allergen-Specific IgE and IgG4 as Biomarkers for Immunologic Changes during Subcutaneous Allergen Immunotherapy

(1) Background: Biomarkers of efficacy for subcutaneous immunotherapy (SCIT) on allergic rhinitis have not been evaluated in details. The present study aims to assess the relevance of measuring of sIgE, sIgG4 and IgE/IgG4 ratio during SCIT in patients with allergic rhinitis; (2) Methods: 20 patients, 13 men and 7 women aged 19 to 58 years, with clinically manifested seasonal and perennial allergic rhinitis were studied. At the initiation and in the end of the three-year course of SCIT serum allergen-specific IgE and IgG4 were measured with ImmunoCAP system. The sIgE/sIgG4 ratio was calculated as a biomarker for immunologic effectiveness; (3) Results: There was a significant increase of sIgG4 antibodies (p < 0.05), while at the end of SCIT for the sIgE levels no significant changes were seen (p > 0.05). Moreover, 90% of patients showed a decrease of the IgE/IgG4 ratio; (4) Conclusions: In most of treated patients with AR, SCIT with Bulgarian allergen products leads to clear immunological changes. After a 3-year of SCIT there is a significant increase in allergen specific IgG4 levels and both decrease of sIgE and IgE/IgG4 ratio. sIgE, sIgG4 and IgE/IgG4 ratio can be used as a substantial biomarker for predicting immunological effectiveness of SCIT.

Immunological Separation of Bioactive Natural Compounds from Crude Drug Extract and Its Application for Cell-Based Studies

In this study, we present a review on a useful approach, namely, immunoaffinity column coupled with monoclonal antibodies (MAbs), to separate natural compounds and its application for cell-based studies. The immunoaffinity column aids in separating the specific target compound from the crude extract. The column capacity was stable even after more than 10 purification cycles of use under the same conditions. After applying the crude extract to the column, the column was washed with washing buffer and eluted with elution buffer. The elution fraction contained the target compound bound to MAb, whereas the washing fraction was the crude extract, which contained all compounds except a group of target compounds; therefore, the washing fraction was referred to as a knockout (KO) crude extract. Cell-based studies using the KO extract revealed the actual effects of the natural compounds in the crude extract. One-step separation of natural compounds using the immunoaffinity column coupled with MAbs may help in determining the potential functions of natural compounds in crude extracts.

Central Nervous System Delivery of Antibodies and Their Single-Domain Antibodies and Variable Fragment Derivatives with Focus on Intranasal Nose to Brain Administration

IgG antibodies are some of the most important biopharmaceutical molecules with a high market volume. In spite of the fact that clinical therapies with antibodies are broadly utilized in oncology, immunology and hematology, their delivery strategies and biodistribution need improvement, their limitations being due to their size and poor ability to penetrate into tissues. In view of their small size, there is a rising interest in derivatives, such as single-domain antibodies and single-chain variable fragments, for clinical diagnostic but also therapeutic applications. Smaller antibody formats combine several benefits for clinical applications and can be manufactured at reduced production costs compared with full-length IgGs. Moreover, such formats have a relevant potential for targeted drug delivery that directs drug cargo to a specific tissue or across the blood–brain barrier. In this review, we give an overview of the challenges for antibody drug delivery in general and focus on intranasal delivery to the central nervous system with antibody formats of different sizes.

Characterization of Anti-Ana o 3 Monoclonal Antibodies and Their Application in Comparing Brazilian Cashew Cultivars

Ana o 3 is an immuno-dominant cashew nut allergen. Four monoclonal antibodies to Ana o 3 (2H5, 6B9C1, 19C9A2, and 5B7F8) were characterized by ELISA and in silico modeling. The 2H5 antibody was the only antibody specific for cashew nut extract. In addition to cashew nut extract, the 6B9C1 and 19C9A2 antibodies recognized pistachio extract, and the 5B7F8 recognized pecan extract. All four antibodies recognized both recombinant Ana o 3.0101 and native Ana o 3. ELISA assays following treatment of purified Ana o 3 with a reducing agent indicated that the 6B9C1 and 19C9A2 antibodies likely recognize conformational epitopes, while the 2H5 and 5B7F8 antibodies likely recognize linear epitopes. In silico modeling predicted distinct epitopes for each of the anti-Ana o 3 antibodies. Screening extracts from 11 Brazilian cashew nut cultivars using all four antibodies showed slight differences in Ana o 3 bindings, demonstrating that these antibodies could identify cultivars with varying allergen content.