Biology subjects

Bunyatov, M.

Publications and source records attributed to Bunyatov, M..

3 recordsLinked to original sources

bioRxiv2025

Engineering affinity-matured variants of an anti-polysialic acid monoclonal antibody with superior cytotoxicity-mediating potency

Monoclonal antibodies (mAbs) that specifically recognize cell surface glycans associated with cancer and infectious disease hold tremendous value for both basic research and clinical applications. However, high-quality anti-glycan mAbs, especially those with sufficiently high affinity and specificity, remain scarce, highlighting the need for protein engineering approaches based on rational design or directed evolution that enable optimization of antigen-binding properties. To this end, we sought to enhance the affinity of a polysialic acid (polySia)-specific antibody called mAb735, which was raised by animal immunization and possesses only modest affinity, using a combination of rational design and directed evolution. The application of these approaches led to the discovery of affinity-matured IgG variants with up to [~]7-fold stronger affinity for polySia relative to the parental antibody. The higher affinity IgG variants were observed to opsonize polySia- positive cancer cells more avidly, which in turn resulted in significantly greater cytotoxicity as determined by both antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. Collectively, these results demonstrate the effective application of both rational and random molecular evolution techniques to an important anti-glycan antibody, providing insights into its carbohydrate recognition while at the same time uncovering variants with greater therapeutic promise due to their enhanced affinity and potency.

bioengineering↗

bioRxiv2022

Synthetic HNK-1 containing glycans provide insight into binding properties of serum antibodies from MAG-neuropathy patients

Anti-myelin-associated glycoprotein (anti-MAG) neuropathy is an autoimmune disease in which IgM autoantibodies target glycoconjugates of peripheral nerves resulting in progressive demyelination. To examine fine specificities of serum IgM autoantibodies and develop a more robust platform for diagnosis and disease monitoring, we describe here a chemoenzymatic approach that readily provided a panel of HNK-1 containing oligosaccharides presented on type 2 oligo-N-acetyl lactosamine (LacNAc) chains typical of glycosphingolipids. The compounds were prepared by a chemoenzymatic strategy in which an oligo-LacNAc structure was assembled enzymatically and then subjected to protecting group manipulation to chemically install a 3-O-sulfate glucuronic acid moiety. The synthetic strategy is highly divergent and made it possible to prepare from key precursors, additional compounds lacking sulfate of HNK-1 and derivatives in which the HNK-1 epitope is replaced by sulfate or sialic acid. The oligosaccharides were printed as a microarray to examine binding specificities of several monoclonal antibodies and serum antibodies of anti-MAG neuropathy patients. Surprisingly, three distinct patient subgroups were identified with variable dependance on the length of the LacNAc chain and sulfation of the glucuronyl moiety. In most cases, a lacto-neohexaose backbone was required for binding indicating the antibodies target corresponding glycosphingolipids. Significance statementA chemoenzymatic strategy is introduced in which a glycan backbone is assembled by glycosyltransferases to give a core oligosaccharide that is subjected to protecting group manipulations and chemical glycosylations to install terminal epitopes. It addresses limitations of enzymatic synthesis when specific glycosyltransferases or glycan-modifying enzymes for terminal epitope synthesis are not readily available. It provided an unprecedented panel of HNK-1 containing oligosaccharides, which was used to develop a glycan microarray that uncovered distinct binding preferences of serum antibodies of anti-MAG patients. The clinical spectrum of IgM monoclonal gammopathy varies substantially and an understanding of binding properties of IgM auto-antibodies will provide opportunities to monitor disease progression and develop personalized treatment options.

biochemistry↗

bioRxiv2021

Functionality of the putative surface glycoproteins of the Wuhan spiny eel influenza virus

A panel of novel influenza-like virus sequences were recently documented in jawless fish, ray-finned fish, and amphibians. Of these, the Wuhan spiny eel influenza virus (WSEIV) was found to phylogenetically cluster with influenza B viruses as a sister clade. Influenza B viruses have been historically documented to circulate only in humans, with certain virus isolates found in harbor seals. It is therefore interesting that a similar virus was potentially found in fish. Here we characterized the functionality and antigenicity of the putative hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins of the WSEIV to better understand this virus and its pandemic potential. Upon functional characterization of NA, we identified that the WSEIV NA-like protein has sialidase activity comparable to B/Malaysia/2506/2004 influenza B virus NA, making it a bona fide neuraminidase that could be inhibited by NA inhibitors. Testing of the functionality of HA was carried out including receptor specificity, stability, and preferential airway protease cleavage and showed very specific binding to monosialic ganglioside 2 (GM2). To probe the degree of conservation of target epitopes, binding of known broadly cross-reactive monoclonal antibodies targeting the influenza B HA and NA, respectively, were assessed through enzyme linked immunosorbent assays against recombinant WSEIV glycoproteins. Human serum samples of patients with antibodies to influenza B viruses were used to determine the cross-reactivity against these novel glycoproteins. Very few monoclonal antibodies - notably including pan NA antibody 1G01 - showed cross-reactivity and reactivity from human sera was basically absent. In summary, we have conducted a functional and antigenic characterization of the glycoproteins of the novel WSEIV to assess if it is indeed a bona fide influenza virus potentially circulating in ray-finned fish.

microbiology↗