Research

With the aging population diseases connected to neurodegeneration are increasing. Thus, the development of effective substances for their prevention and treatment is of utmost priority. However most recently developed medically interesting products, like monoclonal antibodies (mAbs), lack or exhibit poor central nervous system (CNS) penetration. This makes its application difficult. The aim of this proposal is to modulate the biological activity of therapeutically interesting products to efficiently cross the brain-blood-barrier (BBB). An approach that neuroinvasive bacteria use to evade the human immune system and cross the BBB, will be applied. This is achieved by certain sugar polymers, so called polysialic acid (polySia), which forms large negatively-charged hydrodynamic volumes thereby altering bio- chemical, -physical properties of target products. It is hypothesized that target molecules that form micelles (or nano-particle-like structures) and carry polySia with a controlled length, so called low molecular weight (LMW) polySia, are especially effective to cross BBB. To reach the aim a two-tier strategy is applied using a therapeutic monoclonal IgG antibody (mAb) for Alzheimer Disease treatment and the sustainable expression host Nioctiana benthamiana as models. (i) Transfer the LMW-polySia pathway into Nioctiana benthamiana. The approach is based on extensive cross phylum genetics which refers to the transfer of genetic information and the molecular interactions thereof between organisms with large evolutionarily distance. In silico studies suggests that by the co-expression of genetic elements that originate from bacteria, lower and higher eukaryotes in plants allows the assembly of the LMW polySia pathway. (ii) Engineering of mAbs: mAbs are glycoproteins with a single conserved glycosylation site. To design mAbs with altered BBB features two modifications are envisaged (a) enhancing overall glycosylation content by the generation of additional glycosites and (b) design for multimeric IgG formation, to form nanoparticle-like structures. Merging (i) and (ii): Recombinant expression of mutated mAbs in glycoengineered plants. It is expected that recombinant mAbs will carry LMW polySia and form nano-particle-like structures, thereby exhibit efficient CNS penetration. Collectively, by extensive protein and cell engineering products with novel features are generated. The approach may serve as model for other products that need to be delivered to the CNS and generally boosts the engineering of “designer cells” with specified features.

Wider Research Context: Secretory IgA (SIgA) has emerged as a promising candidate for therapeutic interventions owing to its unique structural features including heightened pathogen neutralization efficacy, anti-inflammatory characteristics, and enhanced stability in mucosal secretions. Its interaction with commensal bacteria and mucosal components presents an innovative avenue for novel therapeutic applications. A pivotal determinant of SIgA's unique properties is its extensive glycosylation, yet the glycosylation profile across different tissues and its biological significance remains largely unexplored. Hypotheses: We hypothesize that specific glycan modifications on SIgA are functionally crucial for interactions with components of the mucosa, commensal bacteria and host cell receptors. We aim to fill the knowledge gap on human SIgA glycosylation through a comprehensive analysis of SIgA glycan profiles in different human tissues and subsequent glyco-engineering approaches on recombinant SIgA to elucidate structure-function relationships of distinct glycoforms. Approach: We will elucidate site-specific glycosylation profiles on human SIgA isolated from different tissues, based on which we will employ glyco-engineering tools in the plant-based transient production platform Nicotiana benthamiana a proven and highly suitable system for efficiently producing this intricate and multimeric protein with tailored N- and O-glycans. Generated recombinant SIgA glycoforms will be subjected to extensive biochemical and biophysical characterization as well as in vitro and cell-based binding assays, activation assays and proteolytic stability assessments in mucosal fluids to shed light on the implication of SIgA glycosylation for mucosal immunity and therapeutic development. Innovation: This project innovatively leverages the plant-based transient production platform in Nicotiana benthamiana for glycoengineering, enabling the production of fully assembled and functional SIgA with tailored N- and O-glycans. This approach distinguishes itself from previous studies that relied on heterogeneous glycosylation profiles. The capability to produce large amounts of recombinant human SIgA with homogenous glycoforms allows for unprecedented insights into the structure-function relationships of SIgA, advancing its potential for therapeutic applications. Primary Researchers Involved: Kathrin Göritzer, based at BOKU Vienna as a postdoctoral research fellow (FWF Schrödinger fellowship) has expertise in protein and glyco-engineering of therapeutic antibodies, and has undergone extensive training in immunology. Julian Ma, based at St. George’s University of London is an internationally renowned plant biotechnologist and immunologist.

All living cells are decorated with glycans that are involved in a myriad of essential cellular functions. In mammals, glycoproteins and glycolipids are commonly terminated with the sialic acid Neu5Ac, a negatively charged nine carbon sugar. In contrast to Neu5Ac, the biological functions and immunogenic profiles of a deaminated sialic acid variant called KDN are unknown. The unique features of KDN are (i) resistance to sialidase activity, (ii) termination of polysialic acid chain length, (iii) potential natural decoy against microorganisms that utilize glycoconjugates carrying the host Neu5Ac and, (iv) presence as "oncodevelopmental" antigen marker in various types of cancer. The unique features of KDN and lack of knowledge on its functions contributes to the importance of proposed research. The overall aims of the proposal are to generate KDNylated glycan structures and elucidate the structural and functional impact on selected human glycoproteins. We hypothesize that the coordinated expression of five non-plant enzymes from different species results in the formation of recombinant glycoproteins with KDNylated N-glycans that may have novel biological functions. Addressing the biological function of KDN-containing glycoproteins requires the engineering of the KDN biosynthetic pathway in a suitable host system. Transient expression in plants is a well-established approach for glycoengineering and recombinant protein production and plants are well suited because they lack the interfering Neu5Ac sialic acid biosynthesis pathway. The biosynthetic pathway of KDN will be introduced by transient expression method via agroinfiltration. To optimize expression and activity, a series of expression constructs that carry different combinations of promoter-terminator and subcellular localization signals will be generated and used to produce free KDN, CMP-KDN and KDN-containing N-glycans. The resulting products will be analysed by HPLC and mass spectrometry. In vitro binding assays will be performed to investigate a role of KDN on IgG1 N-glycans in Fcγ-receptor interaction and in vivo experiments will be conducted to investigate the pharmacokinetic profile of KDNylated glycoproteins. In mammalian cells, the biosynthetic pathways of CMP-Neu5Ac and CMP-KDN overlap at different levels. Hence, the presence of a native sialic acid pathway prevents the formation of homogenous KDNylated glycoproteins. Plants produce complex N-glycans but lack a sialic acid pathway. The heterologous expression of the KDN pathway will allow to produce for the first time KDNylated glycoproteins at high homogeneity to study their functional and biochemical characteristics.