Anti-Lea monoclonal antibody SPM 522 recognizes an extended Lea epitope.

Insights into the differential binding characteristics of anti-Lea and anti-LeaLex monoclonal antibodies (mAbs) provide information to develop LeaLex-based cancer immunotherapeutics while avoiding anti-Lea autoimmune reactions. We characterized the epitope recognized by anti-Lea mAb SPM 522. We synthesized the Lea 6-aminohexyl glycoside and report experimental evidence of a minor conformation in solution. The Lea and three other 6-aminohexyl glycosides were conjugated to BSA and titration experiments with SPM 522 show that: 1. SPM 522 binds to LeaLex better than to Lea; 2. the non-reducing Lea galactosyl residue is essential to binding. Competitive ELISA experiments using a panel of tri- to pentasaccharide fragments of LeaLex as well as Lea analogues indicate that: 1. the Lea ß-d-galactosyl α hydrophobic patch is crucial to binding; 2. the Lea fucosyl residue contributes to binding; 3. the Lexd-galactosyl residue also contributes to binding. These results indicate that anti-Lea mAb SPM 522 recognizes the Lea[1,3]-ß-d-Gal tetrasaccharide. We propose that a major recognition element is the extended hydrophobic surface defined by the Lea-ß-d-Gal residue extending to the α faces of the ß-d-GlcNAc and ß-d-Gal residues.

Searching for the Elusive Regulatory Innate Lymphoid Cell.

The complex nature of the innate lymphoid cell (ILC) family and wide range of ILC effector functions has been the focus of intense research. In addition to important roles in host defense, ILCs have central roles in maintaining tissue homeostasis and can promote immune tolerance. Alterations within the microenvironment can impart new functions on ILCs, and can even induce conversion to a distinct ILC family member. Complicating current definitions of ILCs are recent findings of distinct regulatory ILC populations that limit inflammatory responses or recruit other immunosuppressive cells such as regulatory T cells. Whether these populations are distinct ILC family members or rather canonical ILCs that exhibit immunoregulatory functions due to microenvironment signals has been the subject of much debate. In this review, we highlight studies identifying regulatory populations of ILCs that span regulatory NK-like cells, regulatory ILCs, and IL-10-producing ILC2s.

Recognition of Dimeric Lewis X by Anti-Dimeric Lex Antibody SH2.

The carbohydrate antigen dimeric Lewis X (DimLex), which accumulates in colonic and liver adenocarcinomas, is a valuable target to develop anti-cancer therapeutics. Using the native DimLex antigen as a vaccine would elicit an autoimmune response against the Lex antigen found on normal, healthy cells. Thus, we aim to study the immunogenic potential of DimLex and search internal epitopes displayed by DimLex that remain to be recognized by anti-DimLex monoclonal antibodies (mAbs) but no longer possess epitopes recognized by anti-Lex mAbs. In this context, we attempted to map the epitope recognized by anti-DimLex mAb SH2 by titrations and competitive inhibition experiments using oligosaccharide fragments of DimLex as well as Lex analogues. We compare our results with that reported for anti-Lex mAb SH1 and anti-polymeric Lex mAbs 1G5F6 and 291-2G3-A. While SH1 recognizes an epitope localized to the non-reducing end Lex trisaccharide, SH2, 1G5F6, and 291-2G3-A have greater affinity for DimLex conjugates than for Lex conjugates. We show, however, that the Lex trisaccharide is still an important recognition element for SH2, which (like 1G5F6 and 291-2G3-A) makes contacts with all three sugar units of Lex. In contrast to mAb SH1, anti-polymeric Lex mAbs make contact with the GlcNAc acetamido group, suggesting that epitopes extend further from the non-reducing end Lex. Results with SH2 show that this epitope is only recognized when DimLex is presented by glycoconjugates. We have reported that DimLex adopts two conformations around the ß-d-GlcNAc-(1→3)-d-Gal bond connecting the Lex trisaccharides. We propose that only one of these conformations is recognized by SH2 and that this conformation is favored when the hexasaccharide is presented as part of a glycoconjugate such as DimLex-bovine serum albumin (DimLex-BSA). Proper presentation of the oligosaccharide candidate via conjugation to a protein or lipid is essential for the design of an anti-cancer vaccine or immunotherapeutic based on DimLex.

Recognition of Lewis X by Anti-Lex Monoclonal Antibody IG5F6.

mAbs directed toward the Lewis X (Lex) determinant have been shown to display different specificities, depending on the presentation of Lex to the immune system. Of interest is the murine anti-Lex mAb IG5F6, generated against the O chain polysaccharide of Helicobacter pylori that contains polymeric Lex structures. The mAb was found to have a higher affinity for polymeric Lex over monomeric Lex In this study, we explore the recognition of monomeric Lex by IG5F6 using a panel of Lex analogues in which N-acetyl-d-glucosamine, l-fucose, or d-galactose (D-Gal) are replaced with d-glucose and/or l-rhamnose. Our studies show that all analogues were weaker inhibitors than the Lex Ag, indicating that all three residues are essential in the recognition of Lex by mAb IG5F6. We explored the involvement of 4″-OH of d-Gal in the binding with IG5F6 using a panel of 4″-modified Lex analogues. Although the 4″-OH is only involved in a weak polar interaction, we conclude that the D-Gal residue in Lex is primarily involved in aromatic stacking interactions with the Ab binding site. We compared these results to our work with mAb SH1. Although stacking interactions between D-Gal and an aromatic residue was also suggested for SH1, an H-bond involving the 4″-OH was identified that is not found in the binding of IG5F6 to Lex Thus, anti-Lex mAbs SH1 and IG5F6 bind to Lex in different manners, even though the hydrophobic patch displayed by the ß-galactoside in Lex is essential in both cases for their binding to Lex.