Biology subjects

Nedielkov, R.

Publications and source records attributed to Nedielkov, R..

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↗

bioRxiv2025

7β-hydroxysteroid dehydratase Hsh3 eliminates the 7-hydroxy group of the bile salt ursodeoxycholate during degradation by Sphingobium sp. strain Chol11 and other Sphingomonadaceae

Bile salts are steroids with distinctive hydroxylation patterns that are produced and excreted by vertebrates. In contrast to common human bile salts, ursodeoxycholate (UDCA) has a 7-hydroxy group in {beta}-configuration and is used in large amounts for treatment of diverse gastrointestinal diseases. We isolated the 7{beta}-hydroxysteroid dehydratase Hsh3 that is involved in UDCA degradation by Sphingobium sp. strain Chol11. Hsh3 eliminates the 7{beta}-hydroxy group as water, leading to a double bond in the B-ring. This is similar to 7-hydroxysteroid dehydratases in this and other strains, but Hsh3 is evolutionary different from these. Purified Hsh3 accepted steroids with and without side chain as substrates and had minor activity with 7-hydroxy groups. The deletion mutant strain Chol11 {Delta}hsh3 had impacted growth with UDCA and accumulated a novel compound. The compound was identified as 3,5-dihydroxy-H-methyl-hexahydro-5-indene-1-one-propanoate, consisting of steroid rings C and D with a C3-side chain carrying the former 7{beta}-hydroxy group, indicating that Hsh3 activity is important especially for the later stages of bile salt-degradation. Hsh3 homologs were found in other sphingomonads that were also able to degrade UDCA as well as in environmental metagenomes. Thus, Hsh3 adds to the bacterial enzyme repertoire for degrading a variety of differently hydroxylated bile-salts. ImportanceThe bacterial degradation of different bile salts is not only important for the removal of these steroidal compounds from the environment but also harbors interesting enzymes for steroid biotechnology. The 7{beta}-hydroxy bile salt UDCA naturally occurs in high concentration in the feces of black bears and has important pharmaceutical relevance for the treatment of different liver-related diseases, for which it is administered in high and increasing amounts. Additionally, it is present in the bile salt pool of humans in trace amounts. While ursodeoxycholate degradation is environmentally important, the enzyme Hsh3 modifies the hydroxy group that confers the medically relevant properties and thus might be interesting for microbiome analyses and biotechnological applications.

microbiology↗

bioRxiv2023

A flexible loop in the paxillin LIM3 domain mediates direct binding to integrin β3

Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance and haemostasis, but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, focal adhesion assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of focal adhesion sites, with the cytoplasmic tails of the integrin {beta}1 and {beta}3 subunits. The binding interface resides in paxillins LIM3 domain, where based on the NMR structure and functional analyses a flexible, seven amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin {beta}3 compromises cell adhesion and motility. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin {beta}3 function.

cell biology↗

bioRxiv2022

Thiol-mediated Uptake of a Cysteine-containing Nanobody for Anti-Cancer Drug Delivery

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Only a few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that recognizes B cell lymphoma in a thiol-dependent manner. CB2 binding strictly requires the presence of a non-conserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 in a thiol-mediated manner such that the nanobody can be used to deliver cytotoxic agents. Hence, surface thiols can be used as lymphoma biomarkers and targeted by thiol-binding nanobodies. Functionalization of internalizable CB2 is the basis for a range of diagnostic and therapeutic applications of this thiol-binding nanobody. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/497993v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1f47ba9org.highwire.dtl.DTLVardef@1e2bd41org.highwire.dtl.DTLVardef@f7151eorg.highwire.dtl.DTLVardef@18b7681_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisNanobody CB2 specifically binds and internalizes into B cell lymphoma via thiol-based interactions. Functionalized CB2 can be used for complement recruitment or drug delivery to lymphoma cells.

cancer biology↗