Research

Trastuzumab (Tz) is the first therapeutic drug FDA-approved to treat ErbB2+ gastric cancers (GC). ErbB2 undergoes extensive glycosylation, which tightly controls receptor cell surface dynamics and activation. Also, cancer-associated glycosylation (TACAs) at the ErbB2 Tz-binding domain actively drive the resistance of GC cells to Tz cytotoxicity, undermining its clinical efficacy. So far, the structural heterogeneity of ErbB2 glycans has hindered the unequivocal identification and validation of Tz-sensitive/resistant TACAs, and the clinical implementation of glycan-sensitive biomarkers for the prognostic and therapeutic stratification of ErbB2+GC patients. Therefore, the role played by specific ErbB2 TACAs in Tz acquired resistance requires further clarification. This has been holdup by several biotechnological hurdles, including efficient synthesis of tailored glycoconjugates. Plants are well suited for the transient expression of glycoproteins without the need for genetic transformation. In terms of glycosylation, plants offer the advantage of a limited N-glycan processing repertoire, thus enabling a flexible stepwise overexpression of glycosyltransferases required to tailor glycans. We aim to elucidate how distinct ErbB2 glycans tune the Tz binding affinity and function, thus validating them as molecular gatekeepers of Tz therapeutic response in ErbB2+ GC. We hypothesize that plant “synthetic glycans” can be exploited to crack the ErbB2 sugar code underlying molecular processes that lead to GC therapy resistance. We will take advantage of plant’s tolerance for glycoengineering to produce ErbB2 mimicking GC glyco-signatures and identify Tz-sensitive/resistant ErbB2 glycoforms. Glycoform-specific ErbB2-Tz affinity will be orthogonally validated in cancer cell settings by CRISPR/Cas9-based precise glycogene editing. The functionality of ErbB2 TACAs will be comprehensively characterized, including receptor subcellular localization; expression levels; oligomerization capacity and activation/signaling threshold in the presence of Tz. Synthesis of homogenous glycans represents a major bottleneck in structure/function correlation studies. Using plants as bioreactors is a promising innovative and sustainable approach to produce and validate ErbB2 glycoforms to study glycan-dependent interactions. The proposed multivalent strategy will disclose mechanisms underlying resistance of ErbB2+ GC to antibody induced-toxicity and propose selected ErbB2 TACAs as novel stratifying biomarkers in the advanced GC clinical setting. Such studies can guide the design of novel personalized and more efficient targeted therapeutic agents, capable of overcoming glycan-mediated molecular resistance.

The hemicellulose xylan is present in both primary and secondary cell walls and is the major non-cellulosic polysaccharide in industrially important biomass such as wood and grasses. Despite the central role of xylans in development, growth, cell wall strength and biomass resilience, we still know very little about the organisation and distribution of xylan biosynthetic proteins in the Golgi apparatus and how these factors influence the biosynthesis of xylan and the cell wall. To fill this gap, we have cloned Arabidopsis thaliana IRREGULAR XYLEM 9 (AtIRX9), AtIRX10, and AtIRX14, which are involved in the synthesis of the xylan backbone. Transient expression of fluorescent protein fusions in Nicotiana benthamiana showed that only simultaneous expression of AtIRX9, AtIRX10, and AtIRX14 results in robust and efficient Golgi localisation, and co-immunoprecipitation experiments clearly showed interactions between the three proteins, indicating the formation of a heterotrimeric protein complex. We hypothesise that the function of xylan biosynthetic enzymes is regulated by protein-protein interactions and different intra-Golgi localisations. We also hypothesise that AtIRX9, AtIRX10 and AtIRX14 are part of a larger multiprotein xylan synthase complex in the Golgi consisting of proteins with distinct functions in xylan biosynthesis. The aim of this project is (1) to identify the molecular and mechanistic determinants responsible for the localisation of AtIRX9/10/14 in the Golgi and the interactions between these proteins, (2) to investigate the effects of modulation of Golgi localisation and protein-protein interactions on xylan biosynthesis and cell wall composition in transgenic Arabidopsis Irx mutants and (3) to identify the interactome of the Arabidopsis xylan synthase complex in planta. The aim is to find means to modulate xylan biosynthesis and cell wall composition in the model organism A. thaliana.

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.