Immune recruitment or suppression by glycan engineering of endogenous and therapeutic antibodies

Human serum IgG contains multiple glycoforms which exhibit a range of binding properties to effector molecules such as cellular Fc receptors. Emerging knowledge of how the Fc glycans contribute to the antibody structure and effector functions has opened new avenues for the exploitation of defined antibody glycoforms in the treatment of diseases. Here, we review the structure and activity of antibody glycoforms and highlight developments in antibody glycoengineering by both the manipulation of the cellular glycosylation machinery and by chemoenzymatic synthesis. We discuss wide ranging applications of antibody glycoengineering in the treatment of cancer, autoimmunity and inflammation. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

Varied presentation of the Thomsen-Friedenreich disaccharide tumor-associated carbohydrate antigen on gold nanoparticles

Three-dimensional self-assembled monolayers of gold coated with the Thomsen-Friedenreich antigen (TF(ag)) disaccharide (beta-Galp-(1-->3)-GalpNAc) in a variety of presentations have been prepared and characterized. Anomalies in the size distribution of our originally synthesized TF(ag)-bearing nanoparticles as shown in dynamic light scattering experiments prompted us to explore the effect of antigen density on the uniformity of the particles. Gold nanoparticles containing a range of densities 'diluted' with copies of the PEG-thiol spacer unit showed that lower antigen density affords more uniform particles. We also wanted to study the constitution of the actual antigen by synthesizing nanoparticles not only with the linker-extended disaccharide, but also within the context of the surrounding peptide sequence where it may be presented in vivo. The synthesis of TF(ag)-containing glycopeptide thiols based on a mucin peptide repeating unit were prepared, assembled into gold nanoparticles and their physical properties evaluated. These novel multivalent tools should prove extremely useful in exploring the binding properties and immune response to this important carbohydrate antigen.     

The Thomsen-Friedenreich disaccharide as antigen for in vivo tumor targeting with multivalent scFvs

The Thomsen-Friedenreich disaccharide (TF_α) is a promising antigen for tumor immunotargeting, since it is almost exclusively expressed on carcinoma tissues. So far, an obstacle preventing the exploitation of TF for immunotargeting has been the lack of suitable (non-IgM) antibodies with high affinity and specificity. Recently we reported on a novel strategy for generating antibodies toward small uncharged carbohydrates and the generation of recombinant antibodies toward TF. Among them, two multivalent scFv antibodies showed sub-micromolar functional affinities and appeared well suited for immunotargeting. In the present study, the trimeric scFv(1aa) and the tetrameric scFv(0aa) have been further developed for radioimmunotargeting. The scFvs were radiolabeled with ¹¹¹In using DTPA as chelator without losing binding activity or molecular stoichiometry. Binding affinities as high as 1 × 10⁻⁷ M toward TF displayed on living cells were determined. Antibody biodistribution and tumor targeting efficacy were studied in TF-positive human breast cancer (ZR-75-1) bearing mice. TF was successfully targeted in vivo with tumor uptakes of ∼11 and 8% ID/g after 24 h for the trimeric and tetrameric scFv, respectively. These results validate TF as a potent antigen for tumor targeting. The biodistribution of the scFvs was comparable to that reported for IgGs. In contrast to the IgGs, the serum clearance of the scFvs was very fast, which could be an advantage in a therapeutic setting. Furthermore, kidney uptake, which is often critical for small recombinant antibodies labeled with radio-metals, was low with the tetramer (11% ID/g). We conclude that the multimeric anti-TF scFvs are promising candidates to be further developed toward therapeutic application. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Novel immunogenicity of Thomsen-Friedenreich disaccharide obtained by a molecular rotation on its carrier linkage

The alpha-anomeric Galbeta1-3GalNAc, called Thomsen-Friedenreich disaccharide (TFD), is overexpressed in epithelial cancer cells by aberrant O-glycosylation. TFD is also the main ligand of Agaricus bisporus lectin (ABL), a reversible noncytotoxic inhibitor of proliferation of epithelial cell lines. In order to obtain anti-TFD antibody response with a fine carbohydrate-binding specificity similar to that of ABL, we designed an immunogen of TFD with a molecular rotation on its carrier linkage that exposes more GalNAc than Gal, since ABL recognizes GalNAc more than Gal in TFD. The synthesis was accomplished by C-6 oxidation of Gal from TFD or its alpha-benzyl derivative (BzlalphaTFD), followed by reductive amination between the C-6 aldehyde yielded and the available amine of protein. Mice immunized with TFD-KLH (keyhole limpet hemocyanin) or BzlalphaTFD-KLH produced antibodies which were then analyzed by ELISA against several target antigens. Both immunogens raised anti-KLH antibody titers; however, TFD-KLH did not raise anti-TFD antibodies showing low TFD immunogenicity. In contrast, BzlalphaTFD-KLH gave much higher anti-TFD antibody response, indicating that benzyl residue helps improve anti-carbohydrate immune response. When IgG and IgM anti-TFD antibodies were analyzed by competitive ELISA using TFD-related carbohydrates as inhibitors, a high specificity to TFD as well as an enhanced binding to GalNAc over Gal were observed. The axial C-4 hydroxyl group of GalNAc interacted with IgG anti-TFD antibody, as evidenced by the lack of inhibitory activity of GlcNAc in contrast to GalNAc. These findings indicate that the anti-TFD antibodies have fine carbohydrate-binding specificity more similar to ABL than to other TFD-binding proteins that stimulate proliferation of epithelial cell lines.     

Galectin-3 interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes increased cancer cell endothelial adhesion

Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 microg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 +/- 21%, mean +/- S.D.) and 93% (93 +/- 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galbeta1,3 GalNAc-alpha (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-beta1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression.     

Immune response to heavy exertion

Nieman, David C. Immune response to heavyexertion. J. Appl. Physiol. 82(5):1385-1394, 1997.Epidemiological data suggest that enduranceathletes are at increased risk for upper respiratory tract infectionduring periods of heavy training and the 1- to 2-wk period followingrace events. There is growing evidence that, for several hourssubsequent to heavy exertion, several components of both the innate(e.g., natural killer cell activity and neutrophil oxidative burstactivity) and adaptive (e.g., T and B cell function) immune systemexhibit suppressed function. At the same time, plasma pro- andanti-inflammatory cytokines are elevated, in particular interleukin-6-and interleukin-1-receptor antagonist. Various mechanisms explainingthe altered immunity have been explored, including hormone-inducedtrafficking of immune cells and the direct influence of stresshormones, prostaglandin-E₂, cytokines, and other factors. The immune response to heavy exertion istransient, and further research on the mechanisms underlying the immuneresponse to prolonged and intensive endurance exercise is necessarybefore meaningful clinical applications can be drawn. Some attemptshave been made through chemical or nutritional means (e.g.,indomethacin, glutamine, vitamin C, and carbohydrate supplementation) to attenuate immune changes following intensive exercise.

     

Broadening the synthetic potential of disaccharide phosphorylases through enzyme engineering

In recent years, disaccharide phosphorylases have attracted increasing attention as promising biocatalysts for the production of glycosylated compounds. These enzymes make use of a glycosyl phosphate as donor substrate, which is much cheaper than the nucleotide-activated donors required by glycosyl transferases. Unfortunately, the number of available donor specificities is rather limited, and typically only allow the transfer of either a glucosyl or a galactosyl residue. In addition, most phosphorylases have a strong preference for carbohydrate acceptors, and can thus only be used for the synthesis of saccharide chains. The engineering of their substrate specificity thus is of significant value to broaden the range of products that can be obtained. Furthermore, the stability of some phosphorylases will also need to be improved to allow their commercial exploitation in a variety of industrial processes. In this review, several strategies for the engineering of these parameters are discussed and illustrated with some recent successes.     

Immune response of fishes to ciliates

Ciliates are highly evolved protists comprising a phylum of diverse species, many of which are opportunistic or obligate parasites. Ciliates parasitic to fish consist of salt and freshwater forms with endo- or ectoparasitic modes of infection. Some of the more commonly encountered genera include Chilodonella, Brooklynella, Ophryoglenina, Ichthyophthirius, Cryptocaryon, Uronema, Tetrahymena, Epistylus, and Trichodina. Species range from obligate parasites and commensals to opportunistic, facultative forms. Some parasitic ciliates are highly pathogenic and fishes in closed environments such as aquaria and farm ponds are particularly susceptible to high mortalities. Nevertheless, fish have evolved an immune system capable of mounting an effective protective response against parasite challenge. Much of the experimental research on immunity against ciliates has been carried out with Ichthyophthirius multifiliis, on obligate parasite that invades surface epithelia of virtually all freshwater fish species. Interest in the immune response against I. multifiliis stems from the fact that convalescent fish become resistant to subsequent challenge (suggesting the possibility of immunoprophylaxis), and the need to curtail severe losses caused by this parasite in intensively farmed fishes. Furthermore, I. multifiliis has proven to be a useful experimental model because it is amenable to study under laboratory conditions. In this review cellular and humoral factors involved in both innate and acquired immunity against ciliates are covered and include natural killer cells, phagocytic cells, and antibody responses. Current ideas on the mechanisms of antibody-mediated cutaneous immunity against I. multifiliis are discussed and approaches toward the development of vaccines against this and other ciliate parasites are presented.     

Immune response to Encephalitozoon cuniculi infection

Microsporidia are obligate intracellular parasites, which can cause complications in immunocompromised individuals. Very little is known about the host immune response generated against these infectious agents. Encephalitozoon cuniculi is the best studied microsporidian and the protective immune response against this parasite is mediated by cytotoxic CD8(+) T cells.     

Immune response and survival

Immune responses have evolved to defend hosts efficiently against the debilitating effects of parasites on host fitness. However, there are relatively few studies of the efficiency of the immune system in terms of providing hosts with an ability to defend themselves against parasitism. A meta-analysis of the literature on survival of birds in relation to non-specific immune response to challenge with an antigen or other measures of immune function demonstrated a mean effect adjusted for sample size of 0.43 across 12 studies. This observation shows that relatively simple estimates of non-specific immune responses often reliably predict a large and significant amount of variation in survivorship.     

Immune response to stem cells and strategies to induce tolerance

Although recent progress in cardiovascular tissue engineering has generated great expectations for the exploitation of stem cells to restore cardiac form and function, the prospects of a common mass-produced cell resource for clinically viable engineered tissues and organs remain problematic. The refinement of stem cell culture protocols to increase induction of the cardiomyocyte phenotype and the assembly of transplantable vascularized tissue are areas of intense current research, but the problem of immune rejection of heterologous cell type poses perhaps the most significant hurdle to overcome. This article focuses on the potential advantages and problems encountered with various stem cell sources for reconstruction of the damaged or failing myocardium or heart valves and also discusses the need for integrating advances in developmental and stem cell biology, immunology and tissue engineering to achieve the full potential of cardiac tissue engineering. The ultimate goal is to produce 'off-the-shelf' cells and tissues capable of inducing specific immune tolerance.     

Immune response of mice exposed to cis-diamminedichloroplatinum

Summary The effects of cis-diamminedichloroplatinum (CDDP) on lymphoid organs and the immune response of young and older adult mice were studied histologically and by functionally assessing the activity of various subpopulations of immune cells. Young adult mice (6–8 weeks old) treated with 2 mg/kg CDDP mounted an enhanced splenic plaque-forming cell (PFC) response to both sheep erythrocytes, a helper T-cell-dependent antigen (HD), and pneumococcal polysaccharide type III a helper T-cell-independent antigen (HI). Older adult mice (18–22 weeks old) treated in the same way exhibited an equally enhanced PFC response to HD antigen and even a more pronounced response to HI antigen. Treatment of mice with 12 mg/kg CDDP resulted in immunosuppression. Thymus, lymph nodes, and spleen of animals treated with the higher dose of CDDP showed a marked cell depletion from both T and B areas, confirming that the immunosuppression was due to an indiscriminate elimination of both T and B lymphocytes. The immunosuppression and the cell depletion from lymphoid organs were more pronounced in younger mice. Thus, the effects of CDDP on the lymphoid organs and the immune response depend both on the age of the animals and on the dose of the drug. CDDP given in small doses enhances the PFC response, whereas a reduced PFC response is obtained following high-dose treatment. Abbreviations used: CDDP, cis-diamminedichloroplatinum; PFC, plaque-forming cell; HD, helper T-cell dependent; HI, helper T cell-independent; SIII, pneumococcal polysaccharide type III; SRBC, sheep red blood cells; TNP, trinitrophenyl; KLH, keyhole limpet hemocyanin; TNBS, 2, 4, 6-trinitrobenzene sulfonic acid; BBS, borate-buffered saline     

Immune response of rabbits to native G-actins

Actins are highly conserved proteins and are therefore claimed to be not very immunogenic without prior denaturation or chemical modification. We have obtained in rabbits high-titered antibodies to "native" G-actins from chicken and man, and assayed their cross-reaction using an enzyme immunoassay, Western blotting and immunohistochemistry. The antigens differ in their ability to induce antibody formation (chicken gizzard actin [(beta), gamma] greater than chicken skeletal actin [alpha] = human platelet actin [beta, (gamma)]). Antibodies to skeletal actin [alpha] are muscle-specific and mainly directed against the homologous region comprising the N-terminus (residues 1-226). Antibodies to gizzard actin [(beta), gamma] cross-react, to a lesser extent, with the alpha and beta, (gamma) isoforms. They show no regional specificity within the homologous antigen. Antibodies to the tryptic core fragment (residues 69-374) of skeletal actin react with fragments comprising the C-terminal part of muscular actins. Antibodies to platelet actin [beta, (gamma)] cross-react with muscular actins, recognizing not the native, but slightly degraded molecules. Platelet actin induces the formation of high-titered albumin antibodies for hitherto unknown reasons.     

Immune response of fish to parasitic protozoa

Epizootic outbreaks of fish diseases are increasingly common as a result of intensive aquaculture, fish farming and sea ranching. Very few drugs are available for treatment or prophylaxis against fish diseases, and development of such compounds is inhibited by different national regulations governing the use of chemicals in fish for human or animal consumption. Alternative approaches are urgently needed. But although the taxonomy and biology of fish parasites have been extensively studied, relatively little is known about protective immunity in fish and the effects of parasites on the piscine immune system. In this article, Patrick Woo discusses the immune responses of fish to parasitic protozoa, showing that vaccination is a viable control strategy, and stressing the need for a coordinated global research programme on fish diseases.     

Immune response to early and late Histoplasma capsulatum infections

Knowledge of the host response to the intracellular pathogenic fungus Histoplasma capsulatum has increased dramatically. Information has accumulated regarding the cellular and molecular determinants that lead to resolution of both primary and secondary infection. The significance of cytokines and other endogenous soluble mediators to the protective immune response have been analyzed. Moreover, work concerning the relative importance of T cell subsets to protective immunity has been initiated.     

Immune response to a synthetic polysaccharide and protein conjugate

Synthetic polysaccharide (S-PS) containing aglycone-spacer with a free amino group was really alpha 1,6-mannan with Cn approximately 10. S-PS was transformed into isothiocyanate derivative by treating it with thiophosgene and engaged into reaction with amino group of bovine serum albumin (BSA) lysine residues. Rabbits were immunized with S-PS-BSA conjugate and antibodies to S-PS titres were estimated by means of ELISA. S-PS-BSA conjugate was proved to provoke specific anti-polysaccharide antibodies formation in rabbits.