Biology subjects

Seeberger, P.

Publications and source records attributed to Seeberger, P..

4 recordsLinked to original sources

bioRxiv2025

An Integrative Approach to Develop and Characterise Antibodies Against the Cancer Associated Antigen Sialyl Lewis A (CA 19-9)

BackgroundSialyl Lewis A (sLeA), or the CA 19-9 marker, is a tetrasaccharide and a tumour-associated carbohydrate antigen (TACA) overexpressed and abnormally secreted as a serum-borne marker in gastrointestinal malignancies. CA 19-9 is the best validated and only FDA-approved serologic marker clinically used to monitor recurrence, progression, and therapy efficiency in pancreatic ductal adenocarcinoma (PDAC) patients. Due to its altered expression on cancer cells, sLeA is also an attractive target for antibody development. Although recent clinical trials have demonstrated insufficient efficacy of the fully human anti-sLeA 5B1 (MVT-5873) format as a stand-alone drug or an adjuvant therapy in PDAC [1], its safety profile and unique expression in additional malignancies keep CA 19-9 an attractive TACA. Hence, we set out to explore the use of synthetic sLeA to develop novel monoclonal antibodies (mAbs) with improved sLeA recognition and better efficacy. MethodsTwo mAbs targeting sLeA were generated through mice immunisation with synthetic sLeA glycoconjugates, synthetic glycan arrays, and hybridoma technology. We then compared the antigen-binding properties of the newly developed mAbs with the widely used mAb 1116-NS-19- 9 via synthetic glycan arrays, immunohistochemistry (IHC), X-ray crystallography, molecular dynamics (MD) simulation, and Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR) spectroscopy. ResultsThe newly generated mAbs demonstrated improved affinity and specificity for both synthetic and native sLeA, surpassing the performance of the established mAb 1116-NS-19-9. First, synthetic glycan arrays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) assays confirmed superior antigen-binding properties to synthetic sLeA. In particular, the mAb designated GB11 demonstrated markedly enhanced binding to native sLeA ectopically expressed in B16 melanoma cells. To elucidate the structural origin of GB11s improved antigen binding, we conducted high-resolution mapping of the molecular recognition patterns between sLeA and the different antibodies using X-ray crystallography and STD NMR. These analyses revealed subtle yet critical differences in the glycan engagement and identified key structural features underlying GB11s enhanced recognition of sLeA. MD simulations further supported these observations, indicating distinct orientations of sLeA within the binding pockets of each mAb. ConclusionOur results suggest better recognition of the sLeA antigen by the newly generated GB11 antibody and provide a detailed high-resolution elucidation of the molecular interactions behind it. Our study may provide a novel tool with improved theranostic properties against sLeA-overexpressing malignancies.

cancer biology↗

bioRxiv2024

Detection of Antibodies Against the African Parasite Trypanosoma brucei Using Synthetic glycosylphosphatidylinositol oligosaccharide fragments

Trypanosoma brucei (T. brucei) parasites cause two major infectious diseases in Africa: African trypanosomiasis in humans (HAT) and Nagana in animals. Despite the enormous economic and social impact, vaccines and reliable diagnostic measures are still lacking for these diseases. The main obstacle to developing accurate diagnostic methods and an active vaccine is the parasites ability for antigenic variation and impairment of B cell maturation, which prevents the development of a long-lasting, effective immune response. The antigenic variation is sustained by random gene switching, segmental gene conversion, and altered glycosylation states of solvent-exposed regions of the corresponding variant surface glycoproteins (VSG). These glycoproteins use a glycosylphosphatidylinositol (GPI) anchor for attachment to the membrane. GPIs of T. brucei have specific branched structures that are further heterogeneously galactosylated. We synthesized a glycan fragment library containing T. brucei GPIs most prominent structural features and performed an epitope mapping using mice and human sera of infected specimens using glycan microarrays. The studies indicate that in contrast to VSG, T. brucei GPIs are recognized by both short-lived IgM and long-lasting IgG, indicating a specific immune response against GPI structures. These findings enable the development of diagnostic tests based on synthetic antigens for reliable diagnosis of human African trypanosomiasis and Nagana.

biochemistry↗

bioRxiv2019

Glycan size and attachment site location affect electron transfer dissociation (ETD) fragmentation and automated glycopeptide identification

We used a small synthetic glycopeptide library to systematically evaluate the effect of glycosylation site location and glycan size on the efficiency of ETD MS/MS fragmentation and subsequent automated identification. Understanding how the physico-chemical properties of glycopeptides influence glycopeptide fragmentation allows for optimizing fragmentation conditions and software assisted data analyses, which rely on informative fragmentation spectra for subsequent data processing to identify glycopeptides. Often, mis-assignment of glycopeptides occurs due to uncertainties such as failure to produce sufficient peptide backbone fragment ions. Our synthetic glycopeptide library contained glycopeptides differing in glycosylation site position within the peptide as well as glycan size (from the pentasaccharide N-glycan core to fully sialylated, biantennary N-glycans). Different software solutions such as SEQUEST and Amanda were compared for ETD glycopeptide identification. We found that all, glycan size, glycosylation site position within a glycopeptide and individual precursor m/z significantly impacted the number and quality of assignable glycopeptide backbone fragments, and thus the likelihood to be correctly identified in software assisted data analyses.

biochemistry↗

bioRxiv2017

Relevance of host cell surface glycan structure for cell specificity of influenza A virus

Influenza A viruses (IAV) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycan receptors on host cells. HAs receptor specificity towards sialic acid (SA) is well studied and clearly critical for virus infection, but the contribution of the highly complex cellular plasma membrane to the cellular specificity remains elusive. In addition, some studies indicated that other host cell factors such as the epidermal growth factor receptor might contribute to the initial virus-cell contact and further downstream signaling1.\n\nHere we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS) to compare influenza virus receptor specificity with actual host cell binding. Unexpectedly, our study reveals that HAs receptor binding preference does not necessarily reflect virus-cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAV thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.

microbiology↗