Presentation Mode of Glycans Affect Recognition of Human Serum anti-Neu5Gc IgG Antibodies

Recognition of carbohydrates by antibodies can be affected by antigen composition and density. This had been investigated in a variety of controllable multivalent systems using synthetic carbohydrate antigens, yet such effects on anticarbohydrate antibodies in circulating human serum have not been fully addressed thus far. All humans develop a polyclonal and diverse response against carbohydrates containing a nonhuman sialic acid form, N-glycolylneuraminic acid (Neu5Gc). This red meat-derived monosaccharide is incorporated into a diverse collection of human glycans resulting in circulating anti-Neu5Gc antibodies in human sera. Such antibodies can cause exacerbation of diseases mediated by chronic inflammation such as cancer and atherosclerosis. We aimed to evaluate how different presentation modes of Neu5Gc-glycans can affect the detection of anti-Neu5Gc IgGs in human serum. Here, we compare serum IgG recognition of Neu5Gc-containing glycoproteins, glycopeptides, and synthetic glycans. First, Neu5Gc-positive or Neu5Gc-deficient mouse strains were used to generate glycopeptides from serum glycoproteins. Then we developed a reproducible ELISA to screen human sera against Neu5Gc-positive glycopeptides for detection of human serum anti-Neu5Gc IgGs. Finally, we evaluated ELISA screens against glycopeptides in comparison with glycoproteins, as well as against elaborated arrays displaying synthetic Neu5Gc-glycans. Our results demonstrate that the presentation mode and diversity of Neu5Gc-glycans are critical for detection of the full collection of human serum anti-Neu5Gc IgGs

A combined computational-experimental approach to define the structural origin of antibody recognition of sialyl-Tn, a tumor-associated carbohydrate antigen.

Anti-carbohydrate monoclonal antibodies (mAbs) hold great promise as cancer therapeutics and diagnostics. However, their specificity can be mixed, and detailed characterization is problematic, because antibody-glycan complexes are challenging to crystallize. Here, we developed a generalizable approach employing high-throughput techniques for characterizing the structure and specificity of such mAbs, and applied it to the mAb TKH2 developed against the tumor-associated carbohydrate antigen sialyl-Tn (STn). The mAb specificity was defined by apparent KD values determined by quantitative glycan microarray screening. Key residues in the antibody combining site were identified by site-directed mutagenesis, and the glycan-antigen contact surface was defined using saturation transfer difference NMR (STD-NMR). These features were then employed as metrics for selecting the optimal 3D-model of the antibody-glycan complex, out of thousands plausible options generated by automated docking and molecular dynamics simulation. STn-specificity was further validated by computationally screening of the selected antibody 3D-model against the human sialyl-Tn-glycome. This computational-experimental approach would allow rational design of potent antibodies targeting carbohydrates

Characterization of immunogenic Neu5Gc in bioprosthetic heart valves

Background: The two common sialic acids (Sias) in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form N-glycolylneuraminic acid (Neu5Gc). Unlike most mammals, humans cannot synthesize Neu5Gc that is considered foreign and recognized by circulating antibodies. Thus, Neu5Gc is a potential xenogenic carbohydrate antigen in bioprosthetic heart valves (BHV) that tend to deteriorate in time within human patients. Methods: We investigated Neu5Gc expression in non-engineered animal-derived cardiac tissues and in clinically used commercial BHV, and evaluated Neu5Gc immunogenicity on BHV through recognition by human anti-Neu5Gc IgG. Results: Neu5Gc was detected by immunohistochemistry in porcine aortic valves and in porcine and bovine pericardium. Qualitative analysis of Sia linkages revealed Siaa2-3> Siaa2-6 on porcine/bovine pericardium while the opposite in porcine aortic/pulmonary valve cusps. Similarly, six commercial BHV containing either porcine aortic valve or porcine/bovine/equine pericardium revealed Siaa2-3> Siaa2-6 expression. Quantitative analysis of Sia by HPLC showed porcine/bovine pericardium express 4-fold higher Neu5Gc levels compared to the porcine aortic/pulmonary valves, with Neu5Ac at 6-fold over Neu5Gc. Likewise, Neu5Gc was expressed on commercial BHV (186.3 +/- 16.9 pmol Sia/mu g protein), with Neu5Ac at 8-fold over Neu5Gc. Affinity-purified human anti-Neu5Gc IgG showing high specificity toward Neu5Gc-glycans (with no binding to Neu5Ac-glycans) on a glycan microarray, strongly bound to all tested commercial BHV, demonstrating Neu5Gc immune recognition in cardiac xenografts. Conclusions: We conclusively demonstrated Neu5Gc expression in native cardiac tissues, as well as in six commercial BHV. These Neu5Gc xeno-antigens were recognized by human anti-Neu5Gc IgG, supporting their immunogenicity. Altogether, these findings suggest BHV-Neu5Gc/anti-Neu5Gc may play a role in valve deterioration warranting further investigation

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5'monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay.

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5'monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format

Differential Recognition of Diet-Derived Neu5Gc-Neoantigens on Glycan Microarrays by Carbohydrate-Specific Pooled Human IgG and IgA Antibodies

Sialic acids (Sias) cover vertebrate cell surface glycans. N-Acetylneuraminic acid (Neu5Ac) and its hydroxylated form N-glycolylneuraminic acid (Neu5Gc) are common Sia in mammals. Humans cannot synthesize Neu5Gc but accumulate it on cells through red-meat rich diets, generating numerous immunogenic Neu5Gc-neoantigens. Consequently, humans have diverse anti-Neu5Gc antibodies affecting xenotransplantation, cancer, atherosclerosis, and infertility. Anti-Neu5Gc antibodies circulate as IgG, IgM, and IgA isotypes; however, repertoires of the different isotypes in a large population have not been studied yet. Here, we used glycan microarrays to investigate anti-Neu5Gc IgGs and IgAs in intravenous immunoglobulin (IVIG) or pooled human IgA, respectively. Binding patterns on microarrays fabricated with Neu5Gc- and Neu5Ac-glycans, together with inhibition assays, revealed that different IVIG preparations have highly specific anti-Neu5Gc IgG reactivity with closely related repertoires, while IgAs show cross-reactivity against several Neu5Ac-glycans. Such different anti-Neu5Gc IgG/IgA repertoires in individuals could possibly mediate distinctive effects on human diseases

Biomimetic Glyconanoparticle Vaccine for Cancer Immunotherapy

Cancer immunotherapy aims to harness the immune system to combat malignant processes. Transformed cells harbor diverse modifications that lead to formation of neoantigens, including aberrantly expressed cell surface carbohydrates. Targeting tumor-associated carbohydrate antigens (TACA) hold great potential for cancer immunotherapy. N-glycolylneuraminic acid (Neu5Gc) is a dietary non-human immunogenic carbohydrate that accumulates on human cancer cells, thereby generating neoantigens. In mice, passive immunotherapy with anti-Neu5Gc antibodies inhibits growth of Neu5Gc-positive tumors. Here, we designed an active cancer vaccine immunotherapy strategy to target Neu5Gc-positive tumors. We generated biomimetic glyconanoparticles using engineered αGal knockout porcine red blood cells to form nanoghosts (NGs) that either express (NGpos) or lack expression (NGneg) of Neu5Gc-glycoconjugates in their natural context. We demonstrated that optimized immunization of "human-like" Neu5Gc-deficient Cmah-/- mice with NGpos glyconanoparticles induce a strong, diverse and persistent anti-Neu5Gc IgG immune response. The resulting anti-Neu5Gc IgG antibodies were also detected within Neu5Gc-positive tumors and inhibited tumor growth in vivo. Using detailed glycan microarray analysis, we further demonstrate that the kinetics and quality of the immune responses influence the efficacy of the vaccine. These findings reinforce the potential of TACA neoantigens and the dietary non-human sialic acid Neu5Gc, in particular, as immunotherapy targets

Quantitative and qualitative changes in anti‐Neu5Gc antibody response following rabbit anti‐thymocyte IgG induction in kidney allograft recipients

International audienceAntibodies of non-human mammals are glycosylated with carbohydrate antigens, such as galactose-α-1-3-galactose (α-Gal) and N-glycolylneuraminic acid (Neu5Gc). These non-human carbohydrate antigens are highly immunogenic in humans due to loss-of-function mutations of the key genes involved in their synthesis. Such immunogenic carbohydrates are expressed on therapeutic polyclonal rabbit anti-human T-cell IgGs (anti-thymocyte globulin; ATG), the most popular induction treatment in allograft recipients. To decipher the quantitative and qualitative response against these antigens in immunosuppressed patients, particularly against Neu5Gc, which may induce endothelial inflammation in both the graft and the host. We report a prospective study of the antibody response against α-Gal and Neu5Gc-containing glycans following rabbit ATG induction compared to controls. We show a drop in the overall levels of anti-Neu5Gc antibodies at 6 and 12 months post-graft compared to the pre-existing levels due to the major early immunosuppression. However, in contrast, in a cross-sectional study there was a highly significant increase in anti-Neu5Gc IgGs levels at 6 months post-graft in the ATG-treated compared to non-treated patients(P = 0.007), with a clear hierarchy favouring anti-Neu5Gc over anti-Gal response. A sialoglycan microarray analysis revealed that the increased anti-Neu5Gc IgG response was still highly diverse against multiple different Neu5Gc-containing glycans. Furthermore, some of the ATG-treated patients developed a shift in their anti-Neu5Gc IgG repertoire compared with the baseline, recognizing different patterns of Neu5Gc-glycans. In contrast to Gal, Neu5Gc epitopes remain antigenic in severely immunosuppressed patients, who also develop an anti-Neu5Gc repertoire shift. The clinical implications of these observations are discussed

Glycan microarray reveal induced IgGs repertoire shift against a dietary carbohydrate in response to rabbit anti-human thymocyte therapy.

International audienceHumans have circulating antibodies against diverse glycans containing N-glycolylneuraminic acid (Neu5Gc) due to function-loss mutation of the CMAH gene. This xenogenic non-human carbohydrate is abundant in red meat, xenografts and biotherapeutics. Low levels of diet-derived Neu5Gc is also present on normal human endothelial cells, and together with anti-Neu5Gc antibodies could potentially mediate "xenosialitis" chronic-inflammation. Rabbit anti-human thymocyte globulin (ATG) is a drug containing polyclonal IgG glycoproteins commonly used as an immunosuppressant in human transplantation and autoimmune diseases. In type-1 diabetes patients, infusion of Neu5Gc-glycosylated ATG caused increased global anti-Neu5Gc response. Here, for the first time we explore changes in anti-Neu5Gc IgG repertoire following the immunization elicited by ATG, compared with the basal antibodies repertoire that reflect exposure to dietary-Neu5Gc. We used glycan microarrays with multiple Neu5Gc-glycans and controls to elucidate eventual differences in ATG-elicited repertoire, before/after ATG administration and track their kinetics (0, 1, 18 and 24 months). Response of all basal-pre-existing Neu5Gc-specific antibodies rapidly increased. This response peaked at one month post-ATG, with enhanced affinity, then resolved at 18-24 months. Induced-antibodies showed expanded diversity and de-novo recognition of different Neu5Gc-glycans, including endogenous glycolipids, that was further validated by affinity-purified anti-Neu5Gc antibodies from patients' sera. These findings strongly suggest that ATG-induced anti-Neu5Gc IgGs represent a secondary exposure to this dietary carbohydrate-antigen in humans, with immune memory. Given their modified recognition patterns, ATG-evoked anti-Neu5Gc antibodies could potentially mediate biological effects different from pre-existing antibodies