Certain arenaviruses that circulate in rodent populations can cause life-threatening hemorrhagic fevers when they infect humans. Due to their efficient transmission, arenaviruses pose a severe risk ...for outbreaks and might be exploited as biological weapons. Effective countermeasures against these viruses are highly desired. Ideally, a single remedy would be effective against many or even all the pathogenic viruses in this family. However, despite the fact that all pathogenic arenaviruses from South America utilize transferrin receptor 1 (TfR1) as a cellular receptor, their viral glycoproteins are highly diversified, impeding efforts to isolate cross-neutralizing antibodies. Here we address this problem using a rational design approach to target TfR1-tropic arenaviruses with high potency and breadth. The pan-reactive molecule is highly effective against all arenaviruses that were tested, offering a universal therapeutic approach. Our design scheme avoids the shortcomings of previous immunoadhesins and can be used to combat other zoonotic pathogens.
Glycosylation patterns commonly change in cancer, resulting in expression of tumor-associated carbohydrate antigens (TACA). While promising, currently available anti-glycan antibodies are not useful ...for clinical cancer therapy. Here, we show that potent anti-glycan antibodies can be engineered to acquire cancer therapeutic efficacy. We designed yeast surface display to generate and select for therapeutic antibodies against the TACA SLea (CA19−9) in colon and pancreatic cancers. Elite clones showed increased affinity, better specificity, improved binding of human pancreatic and colon cancer cell lines, and increased complement-dependent therapeutic efficacy. Molecular modeling explained the structural basis for improved antibody functionality at the molecular level. These new tools of directed molecular evolution and selection for effective anti-glycan antibodies, provide insights into the mechanisms of cancer therapy targeting glycosylation, and provide major methodological advances that are likely to open up innovative avenues of research in the field of cancer theranostics.
•Neu5Gc is a dietary immunogenic sugar in humans.•It is expressed at low level on human tissues, accumulating mostly on carcinoma.•All humans have circulating anti-Neu5Gc antibodies.•Co-existence of ...Neu5Gc/anti-Neu5Gc spurs chronic inflammation mediated disease.•Concurrently, anti-Neu5Gc antibodies hold potential for novel targeted therapy.
Glycans at the forefront of cells facilitate immune recognition processes. Cancer cells commonly present altered cell surface glycosylation, especially manifested in the expression of sialic acid at the termini of glycolipids and glycoproteins. Although tumor-associated carbohydrate antigens (TACAs) result in expression of altered-self, most such carbohydrates do not elicit strong humoral responses. Various strategies had been devised to elicit increased immunogenicity of such TACA aiming for potent immunotherapeutic antibodies or cancer vaccines. However some carbohydrates are immunogenic in humans and hold potential for novel glycotherapies. N-Glycolylneuraminic acid (Neu5Gc) is a foreign immunogenic sugar in humans originating from the diet (e.g., red meat) and subsequently expressed on the cell surface, especially accumulating on carcinoma. Consequently, the human immune system detects this non-self carbohydrate generating a broad anti-Neu5Gc antibody response. The co-existence of Neu5Gc/anti-Neu5Gc within humans spurs chronic inflammation mediated disease, including cancer. Concurrently, anti-Neu5Gc antibodies hold potential for novel targeted therapy. αGal is another foreign immunogenic carbohydrate antigen in humans and all humans have circulating anti-Gal antibodies. This review aims to describe the immunogenicity of Neu5Gc and its implications for human diseases, highlighting differences and similarities with αGal and its potential for novel targeted theranostics.
Achieving selective inhibition of chemokine activity by structurally well-defined heparan sulfate (HS) or HS mimetic molecules can provide important insights into their roles in individual ...physiological and pathological cellular processes. Here, we report a novel tailor-made HS mimetic, which furnishes an exclusive iduronic acid (IdoA) scaffold with different sulfation patterns and oligosaccharide chain lengths as potential ligands to target chemokines. Notably, highly sulfated-IdoA tetrasaccharide (I-45) exhibited strong binding to CCL2 chemokine thereby blocking CCL2/CCR2-mediated in vitro cancer cell invasion and metastasis. Taken together, IdoA-based HS mimetics offer an alternative HS substrate to generate selective and efficient inhibitors for chemokines and pave the way to a wide range of new therapeutic applications in cancer biology and immunology.
Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We ...hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1-182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in <5% of BHV recipients within 2 years. The levels of both anti-αGal and anti-Neu5Gc IgGs significantly increased one month after BHV implantation. The levels of these IgGs declined thereafter but anti-αGal IgG levels declined significantly faster in control patients compared to BHV recipients. Neu5Gc, anti-Neu5Gc IgG and complement deposition were found in calcified BHVs at much higher levels than in calcified native aortic valves. Moreover, in mice, anti-Neu5Gc antibodies were unable to promote calcium deposition on subcutaneously implanted BHV tissue engineered to lack αGal and Neu5Gc antigens. These results indicate that BHVs manufactured using donor tissues deficient in αGal and Neu5Gc could be less prone to immune-mediated deterioration and have improved durability.
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•CA19-9 is a glycan biomarker of cancer.•Crystal structures of two anti-CA19-9 antibodies were determined in complex with CA19-9.•CA19-9 adopts a preferred low-energy ...conformation.•Recognition of CA19-9 is mediated by intricate networks of polar interactions.•Structure-based design of one antibody resulted with tenfold increase in affinity to CA19-9.
Glycans decorate the cell surface, secreted glycoproteins and glycolipids, and altered glycans are often found in cancers. Despite their high diagnostic and therapeutic potential, however, glycans are polar and flexible molecules that are quite challenging for the development and design of high-affinity binding antibodies. To understand the mechanisms by which glycan neoantigens are specifically recognized by antibodies, we analyze the biomolecular recognition of the tumor-associated carbohydrate antigen CA19-9 by two distinct antibodies using X-ray crystallography. Despite the potential plasticity of glycans and the very different antigen-binding surfaces presented by the antibodies, both structures reveal an essentially identical extended CA19-9 conformer, suggesting that this conformer’s stability selects the antibodies. Starting from the bound structure of one of the antibodies, we use the AbLIFT computational algorithm to design a variant with seven core mutations in the variable domain’s light-heavy chain interface that exhibits tenfold improved affinity for CA19-9. The results reveal strategies used by antibodies to specifically recognize glycan antigens and show how automated antibody-optimization methods may be used to enhance the clinical potential of existing antibodies.
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 K
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.
Humans cannot synthesize N‐glycolylneuraminic acid (Neu5Gc) but dietary Neu5Gc can be absorbed and deposited on endothelial cells (ECs) and diet‐induced anti‐Neu5Gc antibodies (Abs) develop early in ...human life. While the interaction of Neu5Gc and diet‐induced anti‐Neu5Gc Abs occurs in all normal individuals, endothelium activation by elicited anti‐Neu5Gc Abs following a challenge with animal‐derived materials, such as following xenotransplantation, had been postulated. Ten primary human EC preparations were cultured with affinity‐purified anti‐Neu5Gc Abs from human sera obtained before or after exposure to Neu5Gc‐glycosylated rabbit IgGs (elicited Abs). RNAs of each EC preparation stimulated in various conditions by purified Abs were exhaustively sequenced. EC transcriptomic patterns induced by elicited anti‐Neu5Gc Abs, compared with pre‐existing ones, were analyzed. qPCR, cytokines/chemokines release, and apoptosis were tested on some EC preparations. The data showed that anti‐Neu5Gc Abs induced 967 differentially expressed (DE) genes. Most DE genes are shared following EC activation by pre‐existing or anti‐human T‐cell globulin (ATG)‐elicited anti‐Neu5Gc Abs. Compared with pre‐existing anti‐Neu5Gc Abs, which are normal component of ECs environment, elicited anti‐Neu5Gc Abs down‐regulated 66 genes, including master genes of EC function. Furthermore, elicited anti‐Neu5Gc Abs combined with complement‐containing serum down‐regulated most transcripts mobilized by serum alone. Both types of anti‐Neu5Gc Abs‐induced a dose‐ and complement‐dependent release of selected cytokines and chemokines. Altogether, these data show that, compared with pre‐existing anti‐Neu5Gc Abs, ATG‐elicited anti‐Neu5Gc Abs specifically modulate genes related to cytokine responses, MAPkinase cascades, chemotaxis, and integrins and do not skew the EC transcriptome toward a pro‐inflammatory profile in vitro.
The purpose of this research was to determine if Escherichia coli and enterococci in streambed and bank sediments from two urban bayous, Buffalo Bayou and White Oak Bayou, in Houston, TX, USA are a ...significant source of the chronically high levels of these bacteria in the overlying water. The watersheds of the bayous lie within highly urbanized areas of Greater Houston and there is primary recreational contact with the public. Extensive sampling of the watersheds was conducted from 2008 to 2010. Both fecal indicator bacteria were found at ≥104MPNgdry wt.−1 concentrations in the upper 1cm of sediment cores with declines by orders of magnitude at 15 and 30cm sediment horizons and in some cases 60cm, but, nonetheless, indicating that they can remain viable even at depth. No interannual variation was observed. And, there was no correlation with percent organic matter, however there was moderate correlation (R2=0.12; p=0.001) of E. coli with sediment moisture. In sediments, most E. coli and enterococci in Buffalo Bayou (76%) and White Oak Bayou (87.5%) were associated with fine sand grains (60 to 250μm). In the water column, E. coli was associated, in roughly equal percentages, with particle sizes <10, 10–25, 25–63, and ≥63μm (21.9, 25.6, 30.4, and 32.9%, respectively). Enterococci were mostly attached to particle sizes in the ranges of 10–25μm (36.0%) and 25–63μm (31.1%) as well as ≥63μm (37.7%) (p=0.0001). Fingerprinting of E. coli isolates from both bayous with Rep-PCR and the BOX A1R primer was used to demonstrate translocation of sediments from the upper to lower watersheds.
•Streambed and bank sediments were found to be a significant source of E. coli and enterococci bacteria to the water column.•Viable E. coli and enterococci exist as deep as 60cm in sediments.•Sediments dominated by sand contained highest concentrations of fecal indicator bacteria.•DNA fingerprinting analysis challenged the assumption that sediment resuspension only occurs in high flow conditions.•Water quality goals may not be achievable due to an endless supply of fecal indicator bacteria from sediments.
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