Engineered display of ganglioside-sugars on protein elicits a clonally and structurally constrained B cell response

Ganglioside sugars, as Tumour-Associated Carbohydrate Antigens (TACAs), are long-proposed targets for vaccination and therapeutic antibody production, but their self-like character imparts immunorecessive characteristics that classical vaccination approaches have to date failed to overcome. One prominent TACA, the glycan component of ganglioside GM3 (GM3g), is over-expressed on diverse tumours. To probe the limits of glycan tolerance, we used protein editing methods to display GM3g in systematically varied non-native presentation modes by attachment to carrier protein lysine sidechains using diverse chemical linkers. We report here that such presentation creates glycoconjugates that are strongly immunogenic in mice and elicit robust antigen-specific IgG responses specific to GM3g. Characterisation of this response by antigen-specific B cell cloning and phylogenetic and functional analyses suggests that such display enables the engagement of a highly restricted naive B cell class with a defined germline configuration dominated by members of the IGHV2 subgroup. Strikingly, structural analysis reveals that glycan features appear to be recognised primarily by antibody CDRH1/2, and despite the presence of an antigen-specific Th response and B cell somatic hypermutation, we found no evidence of affinity maturation towards the antigen. Together these findings suggest a reach-through model in which glycans, when displayed in non-self formats of sufficient distance from a conjugate backbone, may engage glycan ready V-region motifs encoded in the germline. Structural constraints define why, despite engaging the trisaccharide, antibodies do not bind natively-presented glycans, such as when linked to lipid GM3. Our findings provide an explanation for the long-standing difficulties in raising antibodies reactive with native TACAs, and provide a possible template for rational vaccine design against this and other TACA antigens. HighlightsO_LIGM3g synthetically coupled via a longer, orthogonal (from backbone) glycoconjugate (LOG) presentation format (thioethyl-lysyl-amidine) display elicits high-titre IgG responses in mice. C_LIO_LIThe germinal centre experience of LOG glycoconjugate-specific B cell responses is directly influenced by the protein backbone. C_LIO_LIStructural characterisation of the antibody response to LOGs reveals highly restricted germline-encoded glycan-engaging motifs that mediate GM3g recognition. C_LIO_LIFailure of antibodies to bind the native trisaccharide highlights barriers to be overcome for the rational design of anti-TACA antibodies. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/543556v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b2111org.highwire.dtl.DTLVardef@a81b88org.highwire.dtl.DTLVardef@a78c6aorg.highwire.dtl.DTLVardef@1f3b4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG