A combined computational-experimental approach to define the structural origin of antibody recognition of sialyl-Tn, a tumor-associated carbohydrate antigen

Abstract

Anti-carbohydrate monoclonal antibodies (mAbs) hold great promise as cancer therapeutics and diagnostics. However, their specificity can be mixed, and detailed characterization is problematic, because antibody-glycan complexes are challenging to crystallize. Here, we developed a generalizable approach employing high-throughput techniques for characterizing the structure and specificity of such mAbs, and applied it to the mAb TKH2 developed against the tumor-associated carbohydrate antigen sialyl-Tn (STn). The mAb specificity was defined by apparent KD values determined by quantitative glycan microarray screening. Key residues in the antibody combining site were identified by site-directed mutagenesis, and the glycan-antigen contact surface was defined using saturation transfer difference NMR (STD-NMR). These features were then employed as metrics for selecting the optimal 3D-model of the antibody-glycan complex, out of thousands plausible options generated by automated docking and molecular dynamics simulation. STn-specificity was further validated by computationally screening of the selected antibody 3D-model against the human sialyl-Tn-glycome. This computational-experimental approach would allow rational design of potent antibodies targeting carbohydrates.

Association between Neu5Gc carbohydrate and serum antibodies against it provides the molecular link to cancer: French NutriNet-Santé study

“…The affinity of serum anti-Neu5Gc IgG against diverse Neu5Gc-glycans was analyzed by glycan microarray, as described [ 31 ]. Briefly, slides were developed as above at serial dilutions (a factor of 2) of affinity-purified pooled serum anti-Neu5Gc IgG antibodies ranging at 40–6.1 × 10 −4 ng/μl (266.67–0.033 nM) in PBS/OVA blocking buffer.…”

Synthesis of N-Glycolylneuraminic Acid (Neu5Gc) and Its Glycosides

“…Using efficient OPME sialyltransferase systems with two- or three-enzymes (Figure 8), a diverse array of sialosides including glycosphingolipid glycans, sialylated types 1–5 glycans, and sialyl Tn, containing Neu5Gc (Table 1) as well as sialosides containing different Neu5Gc derivatives including 3F-Neu5Gc, Neu4Ac5Gc, Neu5Gc9Ac, Neu5GcMe, or Neu5GcAc (Table 2) have been synthesized. The obtained compounds have been used to construct sialyl glycan microarrays (28, 33, 34, 42, 83, 150–156), sialoside-protein conjugates (148), and sialidase substrate specificity studies (131, 157–159). Among bacterial sialyltransferases used, Pasteurella multocida sialyltransferase 1 (PmST1) (84) and its single mutant PmST1 M144D with decreased donor hydrolysis and sialidase activities (141) were broadly applied for the synthesis α2–3-linked sialyl oligosaccharides containing Neu5Gc, 3F-Neu5Gc, Neu5Gc9Ac, Neu5GcMe, or Neu5GcAc (77, 83, 84, 89, 157, 160).…”

Directed Evolution of Therapeutic Antibodies Targeting Glycosylation in Cancer

“…Human embryonic kidney 293A cells were used to produce whole native and RA9-mutant antibodies clones from the native and mutated RA9 p3BNC expression plasmids template transfected with polyethylenimine reagent (PEI; Polysciences), as described [ 51 ]. Antibodies were purified using protein A (GE healthcare) and concentrations determined by BCA assay (Pierce).…”