Synthesis of tumor-associated glycopeptide antigens

Carbohydrates and peptides linked together in glycoproteins constitute important components of the molecular communication between cells in multicellular organisms. Cell morphogenesis and tumorigenesis are accompanied by changes in the glycoprotein profiles of the outer cell membranes. Glycopeptide fragments of glycoproteins that have altered structures in tumor cells are of interest as tumor-associated antigens for the distinction between normal cells and tumor cells. In contrast to glycoproteins isolated from biological sources, synthetic glycopeptides are obtained in pure form and exactly specified structures. The methods developed for the synthesis of glycopeptides with tumor-associated antigen structure are outlined in this article by means of a series of typical examples. Beginning with O-glycopeptides of the relatively simple alpha-O-galactosamine-serine/threonine (T(N)-antigen) type, glycopeptide antigens of increasing complexity are described. The review includes syntheses of the saccharide components, the glycosylation reactions to furnish the O-glycosyl amino acid building blocks, their selective C- and N-terminal deprotection and the use of these building blocks for glycopeptide syntheses both in solution and on the solid support. Particular attention is given to glycopeptides containing sialic acid residues, whose syntheses are demanding since reversible protection of the sialic carboxylic group is required. Synthetic methods for the construction of N-glycopeptides carrying the important cell adhesion ligands sialyl Lewis x and sialyl Lewis a antigen are also described. Strategies for the construction of glycopeptides of this type require methods compatible with the presence of the sialic acid residue, as well as with the acid-sensitivity of the fucoside bonds.     

Humoral immunity directed against tumor-associated antigens as potential biomarkers for the early diagnosis of cancer

Over the past decade, it has been demonstrated that cancer is immunogenic, and multiple tumor antigens have been identified in cancer patients. It is now possible to potentially harness the immune response elicited by cancer growth as a potential diagnostic tool. Humoral immunity, or the development of autoantibodies against tumor-associated proteins, may be used as a marker for cancer exposure. Unlike circulating proteins that are shed by bulky tumors, serum autoantibodies are detectable even when antigen expression is minimal. This paper will review the methods used for tumor antigen discovery and overview what is known about autoantibodies targeting common cancer antigens with a focus on breast cancer. Data will be presented modeling the use of tumor antigen associated autoantibodies as a breast cancer diagnostic. The endogenous humoral immune response present in cancer patients may allow the identification of individuals exposed to the malignant transformation of somatic cells.     

Preparation of multifunctional glyconanoparticles as a platform for potential carbohydrate-based anticancer vaccines

A novel platform for anticancer vaccines has been prepared using glyconanotechnology recently developed in our laboratory. Ten different multifunctional gold glyconanoparticles incorporating sialylTn and Lewis(y) antigens, T-cell helper peptides (TT) and glucose in well defined average proportions and with differing density have been synthesised in one step and characterised using NMR and TEM. Size and nature of the linker were crucial to control kinetics of S-Au bond formation and to achieve the desired ligand ratio on the gold clusters. The technology presented here opens the way for tailoring polyvalent anticancer vaccines candidates and drug delivery carriers with defined average chemical composition.     

Autoantibodies against tumor-associated antigens in sputum as biomarkers for lung cancer

Tumor antigens (TAs) can initiate host immune responses and produce TA-associated autoantibody (TAAbs), potential cancer biomarkers. Sputum is directly generated from the upper and lower airways, and thus can be used as a surrogate sample for the diagnosis of lung cancer based on molecular analysis. To develop sputum TAAb biomarkers for the early detection of lung cancer, the leading cause of cancer death, we probed a protein microarray containing more than 9,000 antigens with sputum supernatants of a discovery set of 30 lung cancer patients and 30 cancer-free smokers. Twenty-eight TAs with higher reactivity in sputum of lung cancer cases vs. controls were identified. The diagnostic significance of TAAbs against the TAs was determined by enzyme-linked immunosorbent assays (ELISAs) in sputum of the discovery set and additional 166 lung cancer patients and 213 cancer-free smokers (validation set). Three sputum TAAbs against DDX6, ENO1, and 14–3-3ζ were developed as a biomarker panel with 81% sensitivity and 83% specificity for diagnosis of lung cancer, regardless of stages, locations, and histological types of lung tumors. This study provides the first evidence that sputum TAAbs could be used as biomarkers for the early detection of lung cancer.     

Expression of HIV-1 antigens in plants as potential subunit vaccines

Background

Efforts to improve the efficiency of non-viral gene delivery require a better understanding of delivery kinetics of DNA molecules into clinically relevant cells. Towards this goal, three DNA molecules were employed to investigate the effects of DNA properties on cellular delivery: a circular plasmid DNA (c-DNA), a linearized plasmid DNA (l-DNA) formulated by single-site digestion of c-DNA, and smaller linear gene cassette generated by PCR (pcr-DNA). Four non-viral gene carriers were investigated for DNA delivery: polyethyleneimine (PEI), poly-L-Lysine (PLL), palmitic acid-grafted PLL (PLL-PA), and Lipofectamine-2000?. Particle formation, binding and dissociation characteristics, and DNA uptake by rat bone marrow stromal cells were investigated.

Results

For individual carriers, there was no discernible difference in the morphology of particles formed as a result of carrier complexation with different DNA molecules. With PEI and PLL carriers, no difference was observed in the binding interaction, dissociation characteristics, and DNA uptake among the three DNA molecules. The presence of serum in cell culture media did not significantly affect the DNA delivery by the polymeric carriers, unlike other lipophilic carriers. Using PEI as the carrier, c-DNA was more effective for transgene expression as compared to its linear equivalent (l-DNA) by using the reporter gene for Enhanced Green Fluorescent Protein. pcr-DNA was the least effective despite being delivered into the cells to the same extent.

Conclusion

We conclude that the nature of gene carriers was the primary determinant of cellular delivery of DNA molecules, and circular form of the DNA was more effectively processed for transgene expression.     

Large-scale chemoenzymatic synthesis of blood group and tumor-associated poly-N-acetyllactosamine antigens

Poly-N-acetyllactosamines (pLNs) are common terminal sugars of many N- and O-linked glycan structures present in glycoproteins and glycolipids. Utilizing various glycosyltransferases, we developed new and efficient chemoenzymatic methods for the synthesis of pLNs in gram-scale. Specifically, the use of sialyltransferases and fucosyltransferases enabled us to synthesize and purify 24 blood group and tumor-associated pLN derivatives with alpha-(2-->3)- and alpha-(2-->6)-linked sialic acid, as well as with alpha-(1-->2)- and alpha-(1-->3)-linked fucose. All synthesized derivatives were linked to a short 2-azidoethyl spacer for further modification.     

Direct synthesis of Rev peptide-conjugated gold nanoparticles and their application in cancer therapeutics

We have developed a simple approach for generating peptide-conjugated gold nanoparticles (AuNPs) from the Rev peptide and gold aqueous solution. The peptide functions as both a reducing agent and a capping molecule. AuNPs of various sizes (20-300 nm) and shapes (spheres, triangular plates, and polygons) can be obtained upon modulating the ratio of gold ions to the Rev peptide. Transmission electron microscopy, X-ray diffraction, and UV-vis spectroscopy were utilized to characterize these nanoparticles. Fourier-transform infrared and X-ray photoelectron spectroscopy measurements were performed to investigate chemical interactions between the Rev peptide and AuNPs. Lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays revealed that the Rev peptide-AuNP nanocomposites exhibited exceptionally high cytotoxic effects toward mouse ovarian surface epithelial cell lines, relative to the effects of equal doses of the free Rev peptide. Our study suggests a new way of utilizing biomolecule-conjugated AuNPs as potentially effective anticancer drugs.     

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 potential of DNA vaccination against tumor-associated antigens for antitumor therapy

Conventional treatment approaches for malignant tumors are highly invasive and sometimes have only a palliative effect. Therefore, there is an increasing demand to develop novel, more efficient treatment options. Increased efforts have been made to apply immunomodulatory strategies in antitumor treatment. In recent years, immunizations with naked plasmid DNA encoding tumor-associated antigens have revealed a number of advantages. By DNA vaccination, antigen-specific cellular as well as humoral immune responses can be generated. The induction of specific immune responses directed against antigens expressed in tumor cells and displayed e.g., by MHC class I complexes can inhibit tumor growth and lead to tumor rejection. The improvement of vaccine efficacy has become a critical goal in the development of DNA vaccination as antitumor therapy. The use of different DNA delivery techniques and coadministration of adjuvants including cytokine genes may influence the pattern of specific immune responses induced. This brief review describes recent developments to optimize DNA vaccination against tumor-associated antigens. The prerequisite for a successful antitumor vaccination is breaking tolerance to tumor-associated antigens, which represent "self-antigens." Currently, immunization with xenogeneic DNA to induce immune responses against self-molecules is under intensive investigation. Tumor cells can develop immune escape mechanisms by generation of antigen loss variants, therefore, it may be necessary that DNA vaccines contain more than one tumor antigen. Polyimmunization with a mixture of tumor-associated antigen genes may have a synergistic effect in tumor treatment. The identification of tumor antigens that may serve as targets for DNA immunization has proceeded rapidly. Preclinical studies in animal models are promising that DNA immunization is a potent strategy for mediating antitumor effects in vivo. Thus, DNA vaccines may offer a novel treatment for tumor patients. DNA vaccines may also be useful in the prevention of tumors with genetic predisposition. By DNA vaccination preventing infections, the development of viral-induced tumors may be avoided.     

Liposome encapsulated tumor-associated antigens elicited humoral and cellular immune responses in mice bearing tumor

Chemically induced tumors in mice provide a system to investigate tumor-associated antigens (TAA). The cell surface glycoprotein antigens on such tumor cells have been identified as suitable targets for immune attack. The induction of immune responses against (TAA) in N-nitrosodiethylamine (DEN) exposed mice has been examined. In order to present antigens to the immune system, the liposome was used as vehicle to deliver the TAA. Liposomal-TAA formulation, elicited both humoral and the cellular immune responses, when administered intramuscularly in DEN-exposed mice. Presence of circulatory antibodies against TAA and the induction of cellular responses in immunized mice were monitored using ELISA and in vitro cell proliferation assay of lymphocytes respectively. Specificity of antibody against TAA in immune sera was analysed using immunoblotting technique. Based on these results, it is proposed that the liposome encapsulated TAA may successfully be used to induce humoral and cellular immune responses against tumor.     

Design and synthesis of tetrahydroisoquinoline derivatives as potential multidrug resistance reversal agents in cancer

Exploration for new MDR-modulator utilizing tetrahydroisoquinoline as scaffold disclosed 6,7-dimethoxy-1-(3,4-dimethoxy)benzyl-2-(N-n-octyl-N'-cyano)guanyl-1,2,3,4-tetrahydroisoquinoline (7) as a readily accessible medicinal lead. Compound 7 possessed potent MDR reversal activity in the range of the reference compound verapamil, and had not cardiovascular activity compared to verapamil.     

Fasciola antigens as vaccines against fascioliasis and schistosomiasis

Fascioliasis is an important trematode infection of herbivores worldwide with increasing evidence of prevalence as a disease of humans. Vaccination studies with purified native and recombinant Fasciola antigens suggest that this approach to diminished morbidity and mortality and reduced transmission is a realistic goal. Among the major potential vaccine candidates are fatty acid binding protein (FABP), cysteine (cathepsin) proteases, haemoglobulin, leucine aminopeptidase, and a saposin-like protein. In the case of Fasciola hepatica FABP, cross-reaction and cross-protection against Schistosoma mansoni is an important feature. In addition to protective effects with significant worm burden reductions, some vaccine candidates also have anti-fecundity (smaller flukes), anti-pathology (less liver lesions), and anti-embryonation effects. Optimism is tempered by the fact that fascioliasis in humans is an orphan disease and in need of governmental and foundation support.     

Advances and potential application of gold nanoparticles in nanomedicine

Nanomedicine is an emerging research area which has brought new possibilities and promising applications in image, diagnosis, and treatment. Nanoparticles (NPs) for medicinal purposes can be made of several material types such as silica, carbon, different polymers, and metals as silver, copper, titanium, and gold. Gold NPs (AuNPs) are the most studied and used, mostly due to their characteristics including simple preparation, controllable size and distribution, biocompatibility, good acceptance of surface modifications, and specific surface plasmon resonance (SPR). This study reviews the scientific literature regarding the potential applications of AuNPs in the development of new diagnostic and therapeutic strategies for nanomedicine, including their biomedical use as a drug carrier, as an agent in radio and phototherapy, and bioimaging for image diagnosis. While it becomes clear that much research remains to be done to improve the use of these nanoparticles, with particular concern for safety issues, the evidence from the literature already points to the great potential of AuNPs in nanomedicine.     

Cellular uptake of gold nanoparticles bearing HIV gp120 oligomannosides

Dendritic cells are the most potent of the professional antigen-presenting cells which display a pivotal role in the generation and regulation of adaptive immune responses against HIV-1. The migratory nature of dendritic cells is subverted by HIV-1 to gain access to lymph nodes where viral replication occurs. Dendritic cells express several calcium-dependent C-type lectin receptors including dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN), which constitute a major receptor for HIV-1. DC-SIGN recognizes N-linked high-mannose glycan clusters on HIV gp120 through multivalent and Ca(2+)-dependent protein-carbohydrate interactions. Therefore, mimicking the cluster presentation of oligomannosides from the virus surface is a strategic approach for carbohydrate-based microbicides. We have shown that gold nanoparticles (mannoGNPs) displaying multiple copies of structural motifs (di-, tri-, tetra-, penta-, or heptaoligomanosides) of the N-linked high-mannose glycan of viral gp120 are efficient inhibitors of DC-SIGN-mediated trans-infection of human T cells. We have now prepared the corresponding fluorescent-labeled glyconanoparticles (FITC-mannoGNPs) and studied their uptake by DC-SIGN expressing Burkitt lymphoma cells (Raji DC-SIGN cell line) and monocyte-derived immature dendritic cells (iDCs) by flow cytometry and confocal laser scanning microscopy. We demonstrate that the 1.8 nm oligomannoside coated nanoparticles are endocytosed following both DC-SIGN-dependent and -independent pathways and part of them colocalize with DC-SIGN in early endosomes. The blocking and sequestration of DC-SIGN receptors by mannoGNPs could explain their ability to inhibit HIV-1 trans-infection of human T cells in vitro.     

Characterization of a T-cell clone recognizing idiotypes as tumor-associated antigens

Although heterogeneous T cells recognizing idiotypic determinants have been demonstrated to occur spontaneously in vitro or to be expanded by immunization with antigen or idiotype, their in vitro propagation and cloning was not successful. These previous studies have relied extensively on soluble immunoglobulin to induce proliferation of idiotype-specific T cells. This report describes a unique approach to obtain a stable T-cell clone specific for a monoclonal beta 2-6 fructosan binding myeloma ABPC48 (BALB/c origin), bearing well-defined A48 regulatory idiotopes. Following repeated immunizations with ABPC48 myeloma protein of C.B/R3 mice (H-2d, VHb, CHa), which differ only in the VH locus from BALB/c mice (H-2d, VHa CHa), several stable T-cell clones were obtained after stimulation in vitro with ABPC48 myeloma cells. The proliferation of a T-cell clone A48.B2 was observed with irradiated myeloma cells or hybridomas producing antibodies bearing A48 idiotype encoded by genes deriving from the VH 441-4 family. Proliferation of the clone did not occur with soluble ABPC48 myeloma protein or with Sepharose 4B-bound ABPC48 myeloma protein. Both anti-A48Id and anti-Iad monoclonal antibodies can specifically inhibit the proliferation of this clone when stimulated with ABPC48 myeloma cells. These results demonstrate recognition of idiotypes on B-cell tumours by T cells and implicate the role of class II major histocompatibility complex determinants in this cellular interaction.     

Design,synthesis, and cytotoxic activities of isaindigotone derivatives as potential anti-gastric cancer agents

A series of novel derivatives of isaindigotone, which comes from the root of isaits indinatca Fort, were synthesised (Compound 1–26). Four human gastrointestinal cancer cells (HCT116, PANC-1, SMMC-7721, and AGS) were employed to evaluate the anti-proliferative activity. Among them, Compound 6 displayed the most effective inhibitory activity on AGS cells with an IC₅₀ (50% inhibitory concentration) value of 2.2 μM. The potential mechanism study suggested that Compound 6 induced apoptosis in AGS cells. The collapse of mitochondrial membrane potential (MMP) in AGS cells was proved. In docking analysis, good affinity interaction between Compound 6 and AKT1 was discovered. Treatment of AGS cells with Compound 6 also resulted in significant suppression of PI3K/AKT/mTOR signal pathway. The collapse of MMP and suppression of PI3K/AKT/mTOR signal pathway may be responsible for induction of apoptosis. This derivative Compound 6 could be useful as an underlying anti-tumour agent for treatment of gastric cancer.     

Facile synthesis of Raman active phospholipid gold nanoparticles

Gold nanoparticle-based surface-enhanced Raman scattering (SERS) probes have shown promise for disease detection and diagnosis. To improve their structural and functional stability for in vivo applications, we synthesized a colloidal SERS gold nanoparticle that encapsulates Raman molecules adsorbed on 60 nm gold with a nonthiol phospholipid coating. Transmission electron microscopy and Raman and UV spectroscopy validated its reproducibility and stability. This novel lipid-based SERS probe provides a viable alternative to the PEGylation and silica coating strategies.     

G4-DNA-coated gold nanoparticles: synthesis and assembly

Here, we describe the preparation of stable 15 nm gold nanoparticles (Au-NPs) coated with parallel-stranded G-quadruplexes (G4-DNA), comprising phosphorothioate residues on both sides of the DNA. Phosphorothioate residues located on the surface of the coated particles can anchor them to noncoated ones. Their incubation with more than 20-fold excess of 15 nm citrate-stabilized Au-NPs leads to the formation of flower-shaped structures comprising a central noncoated particle and five to six G-quadruplex-coated ones at the periphery, as revealed by TEM imaging analysis. The absorption band of the structures is shifted toward long wavelengths compared to individual particles not connected to each other. We show a strong dependence of plasmon coupling strength on the length of the DNA connecting Au-NPs.     

Well-defined nanoassemblies using gold nanoparticles bearing specific number of DNA strands

The accurate assembly of nanoparticles into specially designed structures is important for the application of nanoparticle-based materials. Here we report the fabrication of well-defined nanoparticle assemblies via the grouping of gold nanoparticles bearing a specific number of short DNA per particle. Furthermore, we explored various conditions that affect the grouping. Our results show that direct linkage of two nanoparticle-bound DNA without the use of linker DNA yields 80% grouping, which is the highest of all conditions tested. Longer hybridization times and buffer conditions with higher ionic strength also increase grouping formation. These results provide key knowledge that controls the hybridization of nanoparticle-bound DNA for achieving well-defined nanoassemblies with high yield.