Computational Screening of the Human TF-Glycome Provides a Structural Definition for the Specificity of Anti-Tumor Antibody JAA-F11

Abstract

Recombinant antibodies are of profound clinical significance; yet, anti-carbohydrate antibodies are prone to undesirable cross-reactivity with structurally related-glycans. Here we introduce a new technology called Computational Carbohydrate Grafting (CCG), which enables a virtual library of glycans to be assessed for protein binding specificity, and employ it to define the scope and structural origin of the binding specificity of antibody JAA-F11 for glycans containing the Thomsen-Friedenreich (TF) human tumor antigen. A virtual library of the entire human glycome (GLibrary-3D) was constructed, from which 1,182 TF-containing human glycans were identified and assessed for their ability to fit into the antibody combining site. The glycans were categorized into putative binders, or non-binders, on the basis of steric clashes with the antibody surface. The analysis employed a structure of the immune complex, generated by docking the TF-disaccharide (Galβ1-3GalNAcα) into a crystal structure of the JAA-F11 antigen binding fragment, which was shown to be consistent with saturation transfer difference (STD) NMR data. The specificities predicted by CCG were fully consistent with data from experimental glycan array screening, and confirmed that the antibody is selective for the TF-antigen and certain extended core-2 type mucins. Additionally, the CCG analysis identified a limited number of related putative binding motifs, and provided a structural basis for interpreting the specificity. CCG can be utilized to facilitate clinical applications through the determination of the three-dimensional interaction of glycans with proteins, thus augmenting drug and vaccine development techniques that seek to optimize the specificity and affinity of neutralizing proteins, which target glycans associated with diseases including cancer and HIV.

Preclinical Studies with JAA-F11 Anti-Thomsen-Friedenreich Monoclonal Antibody for Human Breast Cancer

“…This is consistent with the crystallography and computational carbohydrate threading data, which show seven tyrosines (the labeling target of the iodogen method) to be in the binding site complementary determining regions [19]. Iodine labeling via the iodogen method sterically inhibited antigen binding.…”

“…Cells producing this antibody were cultured in 5% ultra-low immunoglobulin fetal calf serum (GIBCO, Life Technologies, NY, USA) in Dulbecco's modified Eagle medium with added L-glutamine, sodium pyruvate and nonessential amino acids (Mediatech, VA, USA). JAA-F11 was purified using triple ammonium sulfate precipitation to 33.3% saturation followed by Cibacron Blue 3GA affinity chromatography (Sigma-Aldrich, MO, USA) [19]. [20].…”

The Tetrameric Plant Lectin BanLec Neutralizes HIV through Bidentate Binding to Specific Viral Glycans

“…To investigate whether a single glycan-binding interaction could involve both sites on each monomer, we performed MD simulations (Tessier et al, 2013) to sample sterically allowed configurations of glycan binding. Initially, we performed simulations of a small trisaccharide (Manα1-2Manα1-3Manα) bound to each binding site present on the BanLec monomer, and found that each site had a similar binding strength according to per-residue MM-GBSA (Molecular Mechanics/Generalized Born Surface Area) calculations (Table S1).…”

Applying Pose Clustering and MD Simulations To Eliminate False Positives in Molecular Docking

“…Such a fragment of an oligosaccharide is often referred to as the minimal binding determinant (MBD). 26 In the case of 3TV3 and 3C6S, the MBDs are a disaccharide 66 and pentasaccharide, 67 respectively (Figure 2). Docking of the MBD generally improved the results (Table 3); however, an acceptable pose was only found in two of the five docking experiments for 3TV3, demonstrating the importance of multiple docking experiments.…”

“…Structure-based analyses of antibody–carbohydrate or lectin–carbohydrate interactions offer an alternative means to guide affinity or specificity optimization. 17,25,26 …”