Investigation of Immune Responses to Oxidation, Deamidation, And Isomerization in Therapeutic Antibodies using Preclinical Immunogenicity Risk Assessment Assays.

Product- and process- related critical quality attributes have the potential to impact pharmacokinetics, immunogenicity, potency, and safety of biotherapeutics. Among these critical quality attributes are chemical degradations, specifically oxidation, deamidation, and isomerization. These degradations can be induced by stressors such as light, pH, or temperature; they can also occur naturally under normal conditions. The immunogenicity risk of chemical degradations, particularly in the absence of aggregation, has not been thoroughly understood. In this study, model antibodies with known labile residues were stressed to induce each of the three chemical degradation classes. Aggregate-free and chemically modified antibody species were fractionalized and characterized, followed by testing in standardized and qualified preclinical immunogenicity risk assessment assays for dendritic cell internalization and presentation, monocyte activation, and pre-existing reactivity. Preclinical immunogenicity risk was assessed holistically in vitro based on changes in innate activation risk, CD4 T cell risk, and B cell risk compared to corresponding native antibody. The results of this study suggest an overall moderate increase in immune activation potential for the antibody with isomerization, with only slight increases observed in oxidized and deamidated antibodies. These findings could lend understanding to the immunogenicity risk of chemical degradations in therapeutic antibodies and therefore inform optimization engineering at particular labile residues and risk assessment under the Quality by Design framework.

The Human Leukocyte Antigen Class II Immunopeptidome of the SARS-CoV-2 Spike Glycoprotein.

Precise elucidation of the antigen sequences for T cell immunosurveillance greatly enhances our ability to understand and modulate humoral responses to viral infection or active immunization. Mass spectrometry is used to identify 526 unique sequences from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein extracellular domain in a complex with human leukocyte antigen class II molecules on antigen-presenting cells from a panel of healthy donors selected to represent a majority of allele usage from this highly polymorphic molecule. The identified sequences span the entire spike protein, and several sequences are isolated from a majority of the sampled donors, indicating promiscuous binding. Importantly, many peptides derived from the receptor binding domain used for cell entry are identified. This work represents a precise and comprehensive immunopeptidomic investigation with the SARS-CoV-2 spike glycoprotein and allows detailed analysis of features that may aid vaccine development to end the current coronavirus disease 2019 (COVID-19) pandemic.

Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms.

Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro. All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.

Drosophila Acquires a Long-Lasting Body-Size Memory from Visual Feedback.

Grasping an object or crossing a trench requires the integration of information on the operating distance of our limbs with precise distance estimation. The reach of our hands and step size of our legs are learned by the visual feedback we get during our actions. This implicit knowledge of our peripersonal space is first acquired during infancy but will be continuously updated throughout our whole life [1]. In contrast, body size of holometabolous insects does not change after metamorphosis; nevertheless, they do have to learn their body reaches at least once. The body size of Drosophila imagines can vary by about 15% depending on environmental factors like food quality and temperature [2]. To investigate how flies acquire knowledge about and memorize their body size, we studied their decisions to either refrain from or initiate climbing over gaps exceeding their body size [3]. Naive (dark-reared) flies overestimate their size and have to learn it from the parallax motion of the retinal images of objects in their environment while walking. Naive flies can be trained in a striped arena and manipulated to underestimate their size, but once consolidated, this memory seems to last for a lifetime. Consolidation of this memory is stress sensitive only in the first 2 h after training but cannot be retrieved for the next 12 h. We have identified a set of intrinsic, lateral neurons of the protocerebral bridge of the central complex [4, 5] that depend on dCREB2 transcriptional activity for long-term memory consolidation and maintenance.

m6A modulates neuronal functions and sex determination in Drosophila.

N6-methyladenosine RNA (m6A) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. Here we carry out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. We identify the member of the split ends protein family, Spenito, as a novel bona fide subunit of the methyltransferase complex. We further demonstrate important roles of this complex in neuronal functions and sex determination, and implicate the nuclear YT521-B protein as a main m6A effector in these processes. Altogether, our work substantially extends our knowledge of m6A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal.