Crystal structure of an L chain optimised 14F7 anti-ganglioside Fv suggests a unique tumour-specificity through an unusual H-chain CDR3 architecture

Abstract

Targeted cancer immunotherapy offers increased efficacy concomitantly with reduced side effects. One antibody with promising clinical potential is 14F7, which specifically recognises the NeuGc GM3 ganglioside. This antigen is found in the plasma membrane of a range of tumours, but is essentially absent from healthy human cells. 14F7 can discriminate NeuGc GM3 from the very similar NeuAc GM3, a common component of cell membranes. The molecular basis for this unique specificity is poorly understood. Here we designed and expressed 14F7-derived single-chain Fvs (scFvs), which retained the specificity of the parent antibody. Detailed expression and purification protocols are described as well as the synthesis of the NeuGc GM3 trisaccharide. The most successful scFv construct, which comprises an alternative variable light chain (VLA), allowed structure determination to 2.2 Å resolution. The structure gives insights into the conformation of the important CDR H3 loop and the suspected antigen binding site. Furthermore, the presence of VLA instead of the original VL elucidates how this subdomain indirectly stabilises the CDR H3 loop. The current work may serve as a guideline for the efficient production of scFvs for structure determination.

Insight into glycosphingolipid crypticity: Crystal structure of the anti-tumor antibody 14F7 and recognition of NeuGc GM3 ganglioside

“…Data collection and refinement statistics are summarized in Table 1. The crystal was obtained from the same batch of crystallization setups that earlier yielded the scFv apo-structure (PDB ID: 6FFJ 9 ) and retained P 2 1 symmetry upon soaking, with similar unit cell parameters and four scFv molecules in the asymmetric unit. Two of the four molecules (chains A and B) were well defined by electron density in the CDR regions and could be modeled without breaks, whereas parts of CDR H3 could not be traced in chains C and D. One of the molecules (chain A) contained additional electron density corresponding to the trisaccharide ligand (Figure 1B).…”

“…The monoclonal antibody (mAb) 14F7 is an IgG 1 raised by immunizing a BALB/c mouse with N -glycolyl GM3 (NeuGc GM3) complexed with very low-density lipoproteins (VLDLs) 7 . This antibody is known for its exquisite specificity and high affinity to NeuGc GM3, determined by ELISA to be in the low nanomolar range 7-9 . 14F7 has been used to verify the presence of the NeuGc GM3 in a range of tumors including retinoblastoma 10 , non-small cell lung cancer 11 , colon cancer 12 , breast cancer 7,13 and melanoma 7 .…”

“…The structural basis of the discrimination between NeuGc and NeuAc GM3 has, however, remained elusive. Partial understanding has been gained through the crystal structure of the 14F7 Fab 19 and the more recent structure of a 14F7-derived single-chain variable fragment (scFv) harboring an alternative light chain 9 . Both 14F7 formats feature a long CDR H3 loop, which exhibits key residues for antigen binding.…”

“…B 14F7 scFv light (light blue) and heavy (dark blue) chains (PDB ID: 6S2I, chain A; this work). The 14F7 scFv apo-structure (PDB ID: 6FFJ 9 , chain A) is superimposed in grey. Difference electron density ( m F o - D F c ) for the carbohydrate ligand is shown at 3.0 σ (green mesh).…”

The Structural Complexity and Animal Tissue Distribution of N-Glycolylneuraminic Acid (Neu5Gc)-Terminated Glycans. Implications for Their Immunogenicity in Clinical Xenografting

“…The crystal structure of the Fab fragment of the murine anti-Neu5Gc antibody has been resolved at a 2.2 Å resolution and a molecular model of this fragment in complex with the saccharide moiety of N -glycolyl GM3 ganglioside has been generated (Krengel et al, 2004; Bjerregaard-Andersen et al, 2018).…”