{
  "abstract": "Background Aberrant glycosylation is a feature of many cancers, which present unique glycan signatures relative to normal cells. 1 One such aberration is high mannose (HM) linkages consisting of five to nine mannose residues. In healthy cells this structure is truncated and transformed to complex-type glycans in the endoplasmic reticulum. We have previously described an endoglycosidase, Alfi0894, derived from gut bacteria that cleaves HM.2 Inactivating mutations in the glycan binding pocket of these enzymes lead to HM binding artificial lectins. Here we report the design, production and validation of novel Fc-fusion artificial lectins that bind with high specificity and affinity to cancers that have characteristically high oligomannose residues, such as non-small cell lung cancer (NSCLC).Methods Design, purification and characterization of lectibodies: Alfi0894i or its higher-affinity variant ACP cloned into a pCDNA3.4 vector that had the IgG1 Fc sequence, and expressed in Expi293 cells. It was purified using Protein A affinity chromatography. Proteins were further analyzed for thermal stability and size ELISA: Alfi0894i-Fc or EndoS was coated on 96-well plates overnight and allowed to bind to pooled human serum and binding was detected using an HRP-conjugated anti-IgG antibody Lectibody binding assay: HEK293F cells were grown in suspension and treated with Kifunensine for 48 hours before incubating with serial dilutions of lectibody for one hour followed by incubation with an Fc-specific secondary antibody. Cells were plated and incubated with HM-specific enzymes overnight before being analyzed as described above. Adherent A549 cells were also analyzed similarly on an Agilent Penteon flow cytometer. FcGRIIIa binding assay using surface plasmon resonance: Lectibodies were immobilized to a Protein A chip to around 150 RU and different concentrations of the analyte, FCGRIIa 158V, were allowed to flow over the ligand on a Bioacore X100 instrument.Results The lectibodies were remarkably stable and not prone to aggregation ( figure 1). They exhibited thermal stability and size dispersion similar to Rituximab. Our ELISA assays demonstrate that the there are not pre-existing B-cell epitopes in pooled serum that would cause an immune reaction to Alfi0894i. The HM-expressing variant of HEK293F binds in the nM range, and A549 binds with micromolar affinity. All the lectibodies bind with low uM affinity to the Fc receptor FcGRIIIa, similar to Rituximab.Conclusions We demonstrate the feasibility of using an Fc-fusion artificial lectin that specifically recognizes the HM moiety on cancer cells. This is the first reported gut microbe product to be used as a lectibody.References Oh YJ, Dent MW, Freels AR, Zhou Q, Lebrilla CB, Merchant ML, Matoba N. Antitumor activity of a lectibody targeting cancer-associated high-mannose glycans . Mol Ther. 2022 Apr 6;30(4):1523–1535.Sastre DE, Sultana N, M VASN, Huliciak M, Du J, Cifuente JO, Flowers M, Liu X, Lollar P, Trastoy B, Guerin ME, Sundberg EJ. Human gut microbes express functionally distinct endoglycosidases to metabolize the same N-glycan substrate. Nat Commun. 2024;15(1):5123.Abstract 1153 Figure 1Production and characterization of lectibody",
  "authors": [
    {
      "affiliations": [
        "Emory University, Atlanta, GA, USA"
      ],
      "name": "Adeela Ali"
    },
    {
      "affiliations": [
        "Emory University, Atlanta, GA, USA"
      ],
      "name": "Eric Sundberg"
    }
  ],
  "title": "1153 Human gut microbe-derived artificial lectin as a therapeutic platform for aberrantly glycosylated cancer",
  "uid": "fbec1756-8326-5bfd-bc6e-6d7fbe66eff6"
}