In fact, the two glycolipids only differ by the presence of one additional oxygen atom (H to OH) present in NeuGc GM3 ( Figure 1A). The ability of 14F7 to effectively differentiate between the highly similar NeuGc and NeuAc epitopes is intriguing. NeuGc GM3 is composed of a ceramide tail, buried in the plasma membrane of the cell, and an exposed trisaccharide head group (Neuα2-3Galβ1-4Glcβ) featuring the sialic acid NeuGc at its tip ( Labrada et al. 2008) however, the details of its mode of action are unknown. 14F7 mAb has been reported to kill primary tumor cells by a complement-independent mechanism ( Carr et al. Humanizing the mAb yielded 14F7hT (here referred to as 14F7 mAb), which retained its original ability to induce antibody-dependent cellular cytotoxicity in both human and murine NeuGc GM3-expressing cells ( Fernández-Marrero et al.

2015), non-small cell lung cancer ( Blanco et al. 14F7 has been used to verify the presence of the NeuGc GM3 in a range of tumors including retinoblastoma ( Torbidoni et al. This antibody is known for its exquisite specificity and high affinity to NeuGc GM3, determined by enzyme-linked immunosorbent assay (ELISA) to be in the low nanomolar range ( Carr et al. The monoclonal antibody (mAb) 14F7 is an IgG 1 raised by immunizing a BALB/c mouse with NeuGc GM3 complexed with very low-density lipoproteins ( Carr et al. 2019), NeuGc GM3 is displayed to a larger extent by certain cancer cells and thus represents an attractive TACA. The ganglioside N-glycolyl GM3 (NeuGc GM3) is expressed in most mammals but is absent from healthy adult human cells due to a deletion in the cytidine monophosphate- N-acetylneuraminic acid hydroxylase ( CMAH) gene converting NeuAc to NeuGc ( Chou et al. Immunotherapy offers the possibility of specifically targeting TACAs with high affinity through structure-based engineering of monoclonal antibodies ( Scott et al. This has profound impact on the complexity of engineering in the binding site and provides an excellent example of the importance in understanding the water structure in antibody–antigen interactions.Ĭarbohydrate-antibody interactions, N-glycolyl GM3, protein–carbohydrate interactions, water-mediated antibody specificity, water-mediated interaction, X-ray crystal structure IntroductionĬancer cells differ from healthy cells by aberrant glycosylation patterns, displaying tumor-associated carbohydrate antigens (TACAs) ( Dennis et al. The most intriguing finding, however, was that a water molecule centrally placed in the complementarity-determining region directly mediates the specificity of 14F7 to NeuGc GM3. Modeling analysis and previous mutagenesis data suggest that 14F7 may also bind to an alternative NeuGc GM3 conformation, not observed in the crystal structure. Here we present the 2.3 Å crystal structure of the 14F7 antigen-binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. 14F7 is a monoclonal IgG1 with high clinical potential in cancer immunotherapy as it displays extraordinary specificity for NeuGc GM3, while it does not recognize the very similar, ubiquitous NeuAc GM3. There are exceptions however, such as water's density increasing between 0☌ and 4☌.īelow is a table of units in which density is commonly expressed, as well as the densities of some common materials.Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. Increases in temperature tend to decrease density since the volume will generally increase.

An increase in pressure decreases volume, and always increases density. However, when regarding gases, density is largely affected by temperature and pressure. In the case of solids and liquids, the change in density is typically low. Note that density is also affected by pressure and temperature. It is useful to carefully write out whatever values are being worked with, including units, and perform dimensional analysis to ensure that the final result has units of mass There are many different ways to express density, and not using or converting into the proper units will result in an incorrect value. However, it is important to pay special attention to the units used for density calculations. The calculation of density is quite straightforward.

Density of water gm3