Characterization of the antibody response against NeuGcGM3 ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.     

Altered glycosylation in cancer: A promising target for biomarkers and therapeutics

Glycosylation is a well-regulated cell and microenvironment specific post-translational modification. Several glycosyltransferases and glycosidases orchestrate the addition of defined glycan structures on the proteins and lipids. Recent advances and systemic approaches in glycomics have significantly contributed to a better understanding of instrumental roles of glycans in health and diseases. Emerging research evidence recognized aberrantly glycosylated proteins as the modulators of the malignant phenotype of cancer cells. The Cancer Genome Atlas has identified alterations in the expressions of glycosylation-specific genes that are correlated with cancer progression. However, the mechanistic basis remains poorly explored. Recent researches have shown that specific changes in the glycan structures are associated with 'stemness' and epithelial-to-mesenchymal transition of cancer cells. Moreover, epigenetic changes in the glycosylation pattern make the tumor cells capable of escaping immunosurveillance mechanisms. The deciphering roles of glycans in cancer emphasize that glycans can serve as a source for the development of novel clinical biomarkers. The ability of glycans in intervening various stages of tumor progression and the biosynthetic pathways involved in glycan structures constitute a promising target for cancer therapy. Advances in the knowledge of innovative strategies for identifying the mechanisms of glycan-binding proteins are hoped to hold great potential in cancer therapy. This review discusses the fundamental role of glycans in regulating tumorigenesis and tumor progression and provides insights into the influence of glycans in the current tactics of targeted therapies in the clinical setting.     

Large induces functional glycans in an O-mannosylation dependent manner and targets GlcNAc terminals on alpha-dystroglycan

Alpha-dystroglycan (α-DG) is a ubiquitously expressed receptor for extracellular matrix proteins and some viruses, and plays a pivotal role in a number of pathological events, including cancer progression, muscular dystrophies, and viral infection. The O-glycans on α-DG are essential for its ligand binding, but the biosynthesis of the functional O-glycans remains obscure. The fact that transient overexpression of LARGE, a putative glycosyltransferase, up-regulates the functional glycans on α-DG to mediate its ligand binding implied that overexpression of LARGE may be a novel strategy to treat disorders with hypoglycosylation of α-DG. In this study, we focus on the effects of stable overexpression of Large on α-DG glycosylation in Chinese hamster ovary (CHO) cell and its glycosylation deficient mutants. Surprisingly, stable overexpression of Large in an O-mannosylation null deficient Lec15.2 CHO cells failed to induce the functional glycans on α-DG. Introducing the wild-type DPM2 cDNA, the deficient gene in the Lec15.2 cells, fully restored the Large-induced functional glycosylation, suggesting that Large induces the functional glycans in a DPM2/O-mannosylation dependent manner. Furthermore, stable overexpression of Large can effectively induce functional glycans on N-linked glycans in the Lec8 cells and ldlD cells growing in Gal deficient media, in both of which circumstances galactosylation are deficient. In addition, supplement of Gal to the ldlD cell culture media significantly reduces the amount of functional glycans induced by Large, suggested that galactosylation suppresses Large to induce the functional glycans. Thus our results revealed a mechanism by which Large competes with galactosyltransferase to target GlcNAc terminals to induce the functional glycans on α-DG.     

Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

Protein glycosylation often changes during cancer development, resulting in the expression of cancer-associated carbohydrate antigens. In particular mucins such as MUC1 are subject to these changes. We previously identified an immunodominant Tn-MUC1 (GalNAc-α-MUC1) cancer-specific epitope not covered by immunological tolerance in MUC1 humanized mice and man. The objective of this study was to determine if mouse antibodies to this Tn-MUC1 epitope induce antibody-dependent cellular cytotoxicity (ADCC) pivotal for their potential use in cancer immunotherapy. Binding affinity of mAb 5E5 directed to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis demonstrated high affinity binding (K_D?=?1.7 nM) of mAb 5E5 to its target, Tn-MUC1. Immunolabelling with mAb 5E5 revealed surface expression of the Tn-MUC1 epitope in breast cancer tissue and cell lines, and mAb 5E5 induced ADCC in two human breast cancer cell lines, MCF7 and T47D. Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity suggesting that antibodies targeting glycopeptide epitopes on mucins are strong candidates for cancer-specific immunotherapies.     

Dynamics of immature mAb glycoform secretion during CHO cell culture: An integrated modelling framework

Ensuring consistent glycosylation‐associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy and immunogenicity. However, the interaction between bioprocess conditions, cellular metabolism and the intracellular process of glycosylation remains to be fully understood. To gain further insight into these interactions, we present a novel integrated modelling platform that links dynamic variations in mAb glycosylation with cellular secretory capacity. Two alternative mechanistic representations of how mAb specific productivity (q_p) influences glycosylation are compared. In the first, mAb glycosylation is modulated by the linear velocity with which secretory cargo traverses the Golgi apparatus. In the second, glycosylation is influenced by variations in Golgi volume. Within our modelling framework, both mechanisms accurately reproduce experimentally‐observed dynamic changes in mAb glycosylation. In addition, an optimisation‐based strategy has been developed to estimate the concentration of glycosylation enzymes required to minimise mAb glycoform variability. Our results suggest that the availability of glycosylation machinery relative to cellular secretory capacity may play a crucial role in mAb glycosylation. In the future, the modelling framework presented here may aid in selecting and engineering cell lines that ensure consistent mAb glycosylatio.     

Lectin microarray profiling of metastatic breast cancers

Altered protein glycosylation compared with the disease-free state is a universal feature of cancer cells. It has long been established that distinct glycan structures are associated with specific forms of cancer, but far less is known about the complete array of glycans associated with certain tumors. The cancer glycome has great potential as a source of biomarkers, but progress in this field has been hindered by a lack of available techniques for the elucidation of disease-associated glycosylation. In the present study, lectin microarrays consisting of 45 lectins with different binding preferences covering N- and O-linked glycans were coupled with evanescent-field activated fluorescent detection in the glycomic analysis of primary breast tumors and the serum and urine of patients with metastatic breast cancer. A single 50 μm section of a primary breast tumor or <1 μL of breast cancer patient serum or urine was sufficient to detect glycosylation alterations associated with metastatic breast cancer, as inferred from lectin-binding patterns. The high-throughput, sensitive and relatively simple nature of the simultaneous analysis of N- and O-linked glycosylation following minimal sample preparation and without the need for protein deglycosylation makes the lectin microarray analysis described a valuable tool for discovery phase glycomic profiling.     

Glycan changes: cancer metastasis and anti-cancer vaccines

Complex carbohydrates, which are major components of the cell membrane, perform important functions in cell-cell and cell-extracellular matrix interactions, as well as in signal transduction. They comprise three kinds of biomolecules: glycoproteins, proteoglycans and glycosphingolipids. Recent studies have also shown that glycan changes in malignant cells take a variety of forms and mediate key pathophysiological events during the various stages of tumour progression. Glycosylation changes are universal hallmarks of malignant transformation and tumour progression in human cancer, which take place on the whole cells or some specific molecules. Accordingly, those changes make them prominent candidates for cancer biomarkers in the meantime. This review mainly focuses on the correlation between glycosylation and the metastasis potential of tumour cells from comprehensive aspects to further address the vital roles of glycans in oncogenesising. Moreover, utilizing these glycosylation changes to ward off tumour metastasis by means of anti-adhesion approach or devising anti-cancer vaccine is one of promising targets of future study.     

Impact on N-Glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties

Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for sup-pressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet. The goals of this study were comparison of structural and functional properties of human anti-D polyclonal and monoclonal Abs and assessment of the possibility to manipulate the effector properties of the mAb. N-Glycosylation and particularly the content of nonfucosylated glycans are crucial for affinity of mAb to FcγRIIIA, which plays the key role in the clearance of sensitized cells. We studied and compared glycoprofiles and FcγRIIIA-mediated hemolytic ability of human polyclonal antibodies and anti-D mAbs produced by human B-cell lines, human-rodent heterohybridomas, and a human non-lymphoid cell line PER.C6. Replacement of producing cell line and use of glycosylation modulators can convert an inert mAb into an active one. Nevertheless, rodent cell lines, as well as human non-lymphoid cells, distort natural glycosylation of human IgG and could lead to the loss of immunosuppressive properties. All of the anti-D mAbs secreted by human B-cell lines have a glycoprofile close to human serum IgG. Hence, the constant ratio of IgG glycoforms in human serum is predetermined by glycosylation at the level of the individual antibody-producing cell. The anti-D fraction of polyclonal anti-D immunoglobulin compared to the total human IgG contains more nonfucosylated glycans. Thus, only human trans-formed B-cells are an appropriate source for efficient anti-D mAbs that can imitate the action of polyclonal anti-D IgG.     

Analyzing the dynamic bacterial glycome with a lectin microarray approach

Glycosylation of bacterial cell surfaces is emerging as a critical factor in symbiosis, pathogenesis, cell-cell interactions and immune evasion. The lack of high-throughput analytical tools to examine bacterial glycans has been a major obstacle to the field and has hindered closer examination of the dynamics of carbohydrate variation. We have recently developed a lectin microarray for the analysis of glycoproteins. Herein we present a rapid analytical system based on this technology for the examination of bacterial glycans. The glycosylation pattern observed distinguishes closely related Escherichia coli strains from one another, providing a facile means of fingerprinting bacteria. In addition, dynamic alterations in the carbohydrate coat of a pathogenic E. coli strain are readily observed. The fast evaluation of real-time alterations in surface-carbohydrate epitopes allows examination of the dynamic role of bacterial sugars in response to external stimuli such as the immune system.     

Lung cancer and Toll-like receptors

Lung carcinoma is one of the leading causes of death worldwide. It is a non-immunogenic cancer, resistant to immune surveillance. Toll-like receptors (TLRs) connect the innate to the adaptive immune system. Given that cancerous cells evade the immune system, the activation of TLRs could represent a potential target for cancer therapy. The induction of Th1-like and cytotoxic immunity by TLR signalling could lead to tumour cell death, resulting in tumour regression or arrest. However, basic research and clinical trials revealed that the activation of specific TLRs, such as TLR2, TLR4 and TLR9, do not have any anti-tumour activity in lung carcinoma. Increasing evidence suggests that TLRs are important regulators of tumour biology; however, little is known about their function in lung cancer. Thus, in order to develop new therapeutic approaches, further studies are needed to understand the connection between TLRs and lung cancer progression. This review focuses on the potential mechanisms by which TLR ligands can facilitate or not lung cancer and lung metastases establishment/progression.     

Controlled glycosylation of therapeutic antibodies in plants

Recombinant therapeutic monoclonal antibodies (mAb) can be expressed, assembled, and glycosylated in plants. Transgenic plants, producing anti-rabies mAb and anti-colorectal cancer mAb, were obtained from Agrobacterium-mediated transformation. The heavy chain (HC) of anti-rabies mAb was fused to the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal whereas the HC of anti-colorectal cancer mAb was not fused to the KDEL sequence. Gel release of glycans and detection by high-performance liquid chromatography (HPLC), together with computer assisted analysis and matrix-assisted laser desorption/ionization time-of-flight (MALD-TOF) mass spectrometry, revealed that the plant-derived anti-rabies mAb with KDEL contained mainly oligomannose type N-glycans while the plant-derived anti-colorectal cancer mAb carried mainly biantennary glycans with and without a pentose sugar, that is thought to be xylose. This finding indicates that the KDEL sequence can affect the N-glycosylation processing of antibody in plant cells. The plant-derived mAbs with addition of a KDEL sequence did not contain any of the known antigenic glycan epitopes that are frequently found in other plant glycans or in mammalian-derived mAbs. The altered glycosylation on both plant-derived mAbs did not affect the activities that are required for therapy. These results indicate that plant genetic engineering could provide an effective and inexpensive means to control the glycosylation of therapeutic proteins such as mAbs, by the addition of a KDEL signal as a regulatory element.     

Lonely killers

The majority of the most effective monoclonal antibodies (mAbs) currently in the clinics bind to cancer or immune cells. Classic mechanisms of cell killing by therapeutic mAbs include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and induction of apoptosis by engagement of specific cell ligands. A few reports have described mAbs whose cytotoxic activity is Fc-independent and that do not induce the morphological and biochemical changes associated with the apoptosis-type of cell death. Even fewer works describe mAbs able to directly induce membrane lesions. Here, we discuss the available data on those molecules and their cell killing activity, with particular attention to the case of a mAb specific for the tumor-associated N-glycolyl (Neu5Gc)-GM3 ganglioside (GM3(Neu5Gc)). Some similarities are found in the cell death pathways triggered by these mAbs, but data are not abundant. We conclude that the usefulness of mAbs with a direct cytotoxic activity for immunotherapeutic strategies deserves deeper research.     

Immunogenic cell death in cancer therapy: Present and emerging inducers

In the tumour microenvironment (TME), immunogenic cell death (ICD) plays a major role in stimulating the dysfunctional antitumour immune system. Chronic exposure of damage‐associated molecular patterns (DAMPs) attracts receptors and ligands on dendritic cells (DCs) and activates immature DCs to transition to a mature phenotype, which promotes the processing of phagocytic cargo in DCs and accelerates the engulfment of antigenic components by DCs. Consequently, via antigen presentation, DCs stimulate specific T cell responses that kill more cancer cells. The induction of ICD eventually results in long‐lasting protective antitumour immunity. Through the exploration of ICD inducers, recent studies have shown that there are many novel modalities with the ability to induce immunogenic cancer cell death. In this review, we mainly discussed and summarized the emerging methods for inducing immunogenic cancer cell death. Concepts and molecular mechanisms relevant to antitumour effects of ICD are also briefly discussed.     

The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.     

Cancer stem cell marker glycosylation: Nature,function and significance

Glycans are essential for the maintenance of normal biological function, with alterations in glycan expression being a hallmark of cancer. Cancer stem cells (CSCs) are a subset of cells within a tumour capable of self-renewal, cellular differentiation and resistances to conventional therapies. As is the case with stem cells, marker proteins present on the cell surface are frequently used to identify and enrich CSCs, with the expression of these markers statistical correlating with the likelihood of cancer recurrence and overall patient survival. As such CSC markers are of high clinical relevance. The majority of markers currently used to identify CSC populations are glycoproteins, and although the diverse biological roles for many of these markers are known, the nature and function of the glycan moiety on these glycoproteins remains to be fully elucidated. This mini-review summarises our current knowledge regarding the types and extent of CSC marker glycosylation, and the various roles that these glycans play in CSC biology, including in mediating cell adhesion, metastasis, evading apoptosis, tear shear resistance, tumour growth, maintaining pluripotency, self-renewal, trafficking, maintaining stability, maintaining enzymatic activity and aiding epithelial mesenchymal transitioning. Given that CSCs markers have multiple diverse biological functions, and are potentially of significant diagnostic and therapeutic benefit the search for new markers that are uniquely expressed on CSCs is vital to selectively target/identify this subset of cancer cells. As such we have also outlined how high-throughput lectin microarrays can be used to successfully profile the glycosylation status of CSC and to identify glyco-markers unique to CSCs.     

Lonely killers: Effector cell- and complement-independent non-proapoptotic cytotoxic antibodies inducing membrane lesions

The majority of the most effective monoclonal antibodies (mAbs) currently in the clinics bind to cancer or immune cells. Classic mechanisms of cell killing by therapeutic mAbs include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and induction of apoptosis by engagement of specific cell ligands. A few reports have described mAbs whose cytotoxic activity is Fc-independent and that do not induce the morphological and biochemical changes associated with the apoptosis-type of cell death. Even fewer works describe mAbs able to directly induce membrane lesions. Here, we discuss the available data on those molecules and their cell killing activity, with particular attention to the case of a mAb specific for the tumor-associated N-glycolyl (Neu5Gc)-GM3 ganglioside [GM3(Neu5Gc)]. Some similarities are found in the cell death pathways triggered by these mAbs, but data are not abundant. We conclude that the usefulness of mAbs with a direct cytotoxic activity for immunotherapeutic strategies deserves deeper research.Key words: cytotoxicity, therapeutic antibody, necrosis, membrane lesion, cell death     

Dll4-Notch Signalling Blockade Synergizes Combined Ultrasound-Stimulated Microbubble and Radiation Therapy in Human Colon Cancer Xenografts

Tumour vasculature acts as an essential lifeline for tumour progression and facilitates metastatic spread. Novel vascular targeting strategies aiming to sustain vascular shutdown could potentially induce substantial damage, resulting in a significant tumour growth delay. We investigated the combination of two novel complementary vascular targeting agents with radiation therapy in a strategy aiming to sustain vascular disruption. Experiments were carried out with delta-like ligand 4 (Dll4) blockade (angiogenesis deregulator) treatment administered in combination with a radiation-based vascular destruction treatment in a highly aggressive well-perfused colon cancer tumour line implanted in female athymic nude mice. Tumours were treated with permutations of radiation, ultrasound-stimulated microbubbles (USMB) and Dll4 monoclonal antibody (mAb). Tumour vascular response was assessed with three-dimensional power Doppler ultrasound to measure active flow and immunohistochemistry. Tumour response was assessed with histochemical assays and longitudinal measurements of tumour volume. Our results suggest a significant tumour response in animals treated with USMB combined with radiation, and Dll4 mAb, leading to a synergistic tumour growth delay of up to 24 days. This is likely linked to rapid cell death within the tumour and a sustained tumour vascular shutdown. We conclude that the triple combination treatments cause a vascular shutdown followed by a sustained inhibition of angiogenesis and tumour cell death, leading to a rapid tumour vascular-based ‘collapse’ and a significant tumour growth delay.     

Anti-tumor immunity provided by a synthetic multiple antigenic glycopeptide displaying a tri-Tn glycotope

In many cancer cells the alteration of glycosylation processes leads to the expression of cryptic carbohydrate moieties, which make them good targets for immune intervention. Identification of cancer-associated glycotopes as well as progress in chemical synthesis have opened up the way for the development of fully synthetic immunogens that can induce anti-saccharide immune responses. Here, we synthesized a dendrimeric multiple antigenic glycopeptide (MAG) containing the Tn Ag O:-linked to a CD4(+) T cell epitope. This MAG is based on three consecutive Tn moieties (tri-Tn) corresponding to the glycotope recognized by an mAb (MLS 128) produced against the LS180 colon carcinoma cell line. The Abs induced by this MAG recognized murine and human tumor cell lines expressing the Tn Ag. Prophylactic vaccination using MAG provided protection of mice against tumor challenge. When used in active specific immunotherapy, the MAG carrying the tri-Tn glycotope was much more efficient than the mono-Tn analogue in promoting the survival of tumor-bearing mice. Furthermore, in active specific immunotherapy, a linear glycopeptide carrying two copies of the tri-Tn glycotope was shown to be poorly efficient compared with the dendrimeric MAG. Therefore, both the clustering of carbohydrate Ags and the way they are displayed seem to be important parameters for stimulating efficient anti-saccharide immune responses.     

Targeted glycoproteomic analysis reveals that kappa-5 is a major, uniquely glycosylated component of Schistosoma mansoni egg antigens

Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAcβ1-4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Galβ1-4(Fucα1-3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity.