Introduction
Under the inspiration of the Warburg effect, approaches of anticancer drugs founded on glycoconjugation are steadily acquiring significant attention from the scientific community. Comparing normal tissues with human and animal tumors, alteration in carbohydrate structures has been found prior to and after transformation by oncogenic agents. This includes non-metastatic and metastatic tumor cells, modified non-tumorigenic and tumorigenic cells, low- and high-metastatic variants, and tumor cells before and after the introduction of differentiation to a phenotype that is less malignant. The identified change in glycosaminoglycans, glycolipids, and glycoproteins have been studied and their structures illustrated by various studies over the years (Hossain & Andreana, 2019). Both changes in glycosyltransferase and nucleotide sugar biosynthesis are linked to these alterations. This report discusses Tumor Carbohydrate Associated Antigen (TACAs), Thomsen–Friedenreich (TF) antigen, synthesis of the TF antigen, comparing the TF antigen to a normal cell, and aptamers and Selex as biomarkers for tumor-carbohydrate associated antigen. Indeed, there has been notable progress over the years in developing carbohydrate-based, synthetic antitumor vaccines to aid in improving immune responses aimed at targeting the explicit antigens.
Tumor Carbohydrate Associated Antigen (TACAs)
A common phenotypic modification of cancer cells, referred to as aberrant glycosylation, arises from a bunched arrangement on the surface of a cell, or through altered synthesis. Carbohydrate moieties that are over-expressed or reformed on the cell membrane form a major tumor characteristic (Hutchins et al., 2017). Therefore, cancer cells are easily distinguished from others by the fact that, on their surface, they display abnormal levels and kinds of carbohydrate structures. Tumor-associated carbohydrate antigens (TACAs) refer to these unusual arrangements, and they play a vital role in metastasis and tumor invasion. Their presence is frequently connected with the poor survival rates of extracellular tumor-specific carbohydrate antigens expressed by cancer cells of different origins due to abnormal glycosylation.
Various cases related the changes in malformed cells to grow, but not transformation. Others linked the altered glycoconjugates to tumor-associated carbohydrate antigens, though evidence has been presented to prove that cell surface glycoconjugates may at times function in growth control (Hossain & Andreana, 2019). Transformed carbohydrate structures also can act as receptors for growth endorsing factors and are accountable for altered growth control. Current studies with monoclonal antibodies have indicated that the majority of antibodies which recognize tumor-associated antigens can detect altered carbohydrate structures. The mechanism by which the immune system is capable of recognizing these structures is put into consideration, and exploration of immune recognition of the same alterations takes place.
TACAs with an overexpression on the tumor cells’ surface is acknowledged as the biomarkers for cancer detection and are accorded priority in the creation of modern carbohydrate-based anti-cancer vaccines. They are known to mediate significant signaling effects which trigger their functions in tumor biology (Hutchins et al., 2017). Owing to the fact that TACAs are conveyed on glycolipids and glycoproteins, using anti-TACA treatment has been an attractive concept in combating cancer. These glycoproteins are identified to help in regulating multiple cellular pathways, pointing out the proliferative machinery of malevolent cells. TACAs have been put into consideration as auspicious targets for the designing of anticancer vaccines (Hutchins et al., 2017). However, carbohydrates alone are only known to induce poor immunogenicity, the reason being that they cannot evoke T-cell-dependent immune reactions, which is usually critical for cancer therapy. Additionally, immunosuppression and immune tolerance can be induced easily by the use of TACAs which occur naturally as antigens, due to the endogenous property that they contain.
Neolactoseries antigen Lewis Y (LeY) and ganglioside GD2 are among the monoclonal antibodies (mAbs), directed to TACAs that are known to arbitrate cell survival signals. Once directed to TACAs, the two hinder cell signaling thereby affecting the existence of a cell directly (Hutchins et al., 2017). Nonetheless, the above oncotargets have been approved and some are in clinical trials. Inducing antibodies to the antigens and other associated types is thus of clinical benefit because it provides sustained immunity, which helps in inhibiting metastatic outgrowth. In immunotherapy development, the usefulness of TACAs has been restricted severely by immune tolerance to abnormal carbohydrate structures, hence, multiple approaches are required to direct the immune reaction. Potential TACA-directed vaccines have been developed, grounded on carbohydrate-mimetic peptides (CMPs) that are capable of inducing cellular and anti-tumor-reactive humoral reactions in mice. The CMPs are normally Pan-immunogens, designed to prompt antibodies that can respond to multiple TACAs when they are immunized with a single agent.
One of them (the CMPs), has been moved with the arrangement WRYTAPVHLGDG (known as P10s), joined with the Pan-T-cell epitope PADRE, during the initial stages of a clinical trial of breast cancer Stage IV issues. This CMP was intended to imitate and prompt reactions to the LeY and the ganglioside GD2 antigens (Hutchins et al., 2017). Specifically, LeY consists of a controlled tissue distribution owing to the fact that it is a fetal antigen. Originally, it was established to be overexpressed on a vast proportion of epithelial-derived tumors, as well as lung, colon, prostate, ovary, and breast-originated tumors. Its antigen promotes cancer cells’ metastasis and incursion, and its great manifestation is correlated with a decreased occurrence of lymph node-negative breast carcinomas. This antigen is supposed to aid in regulating the appearance of cell cycle-related aspects via PI3K/Akt and ERK/MAPK gesticulating pathways to enhance cell propagation. The expression from this antigen is thus considered to be linked with drug resistance.
Just as LeY antigen, anti-GD2 antibodies have the ability to down moderate PI3K/Akt signaling pathways. Since it is a possible target for anti-tumor immunotherapy, the antigen is perfect following its high expression on numerous types of tumors, and the limited expression on normal tissue. In prioritizing the best cancer antigens, the National Cancer Institute (NCI) pilot ranks GD2 #12 over 75 among the possible targets for cancer therapy. Actually, the antigen is ranked #6 when considering antigens that can be targeted directly on the cell surface or in circulation (Hutchinset al., 2017). Ganglioside is conveyed in the breast and on other cancer stem cells. In support of anti-GD2 immunotherapeutic methodologies for the management of breast cancer, the GD2/c-Met axis was discovered to be involved negatively in estrogen receptor (ER) of breast cancer. Its aggressiveness is strong in subgroups of this cancer including metaplastic and triple-negative variants (Hutchins et al., 2017). The b-series, including GD3/GD3, are usually confirmed at high levels in the sera of subjects of breast cancer.
Inducing the antibodies to GD2 or LeY can greatly interfere with the signals of cancer cell survival. It is certain that TACA bunching or density can have a significant effect on being recognized by antibodies. The physiognomies of carbohydrate epitopes that appear on tumor cells have been identified to be a factor that governs the immune recognized, antibody-mediated, and targeted by carbohydrate antigens in cancer immunotherapy. The interaction between density-dependent and lectin-glycan is an exemplar of the provisional guidelines by posttranslational alterations (Hutchins et al., 2017). Antibodies that react with TACAs are proposed to be discriminative for bunched TACA even when normal tissues are present. TACAs which result either from incomplete synthesis, regulated circulation on the normal cell surface, or biosynthesis accrue in high concentration or bunched conformation, probably in new configurations comparative to glycans conveyed on normal tissue (Hutchins et al., 2017). These may be found at the tumor cell surface and are likely to selectively lack immune arbitrated tissue destruction even though antibodies have the ability to muddle the normal tissue.
If the signaling mechanisms linked to TACAs expression are interfered with, tumor growth may stop, and the development of metastases may be prevented. As part of both immune surveillance and monoclonal antibodies, TACA-reactive antibodies are usually said to be proapoptotic. Ganglioside GD2 and LeY antigens are capable of regulating the processes of signaling in various ways that advance the progression of cancer. In essence, the antibodies’ polyspecific nature is exploited in developing P10s as a Pan-immunogen, which is reactive with lectins and multiple anti-TACA. The induction of proapoptotic, polyreactive antibodies is a helpful feature in the approaches of cancer immunotherapy (Hutchins et al., 2017). Inducing these antibodies makes the subjects of cancer survive for a long time because they are clinically associated with the same. Consequently, P10s are developed for medical testing of inoculation of breast cancer subjects who are at high risk.
TF Antigen
Thomsen–Friedenreich (TF) antigen is usually a disaccharide which is existing on the cell surfaces in a mysterious form enclosed by N-acetylneuraminic acid moieties and is circulated in various types of cancer. The antigen epitomizes a tumor-associated molecule, presumed to be among the rare chemically definite antigens which show a confirmed association with malignancy. With an aim of analyzing the function of the carbohydrate structure Gal-GalNAc for gastral tumors, MCF-7 breast tumors with Balb/c mice cells were injected, and synthetic Gal-GalNAc was associated with BSA carrier. From the result, a monoclonal antibody that recognizes the TF antigen was established (Sindrewicz et al., 2016). The early biochemical study discovered that the T-determinant can be detected in the high-molecular-weight range (about 1000 kD), signifying that the Gal-GalNAc epitope is commonly present on mucin-like glycoproteins. Thus, the tumor constraint of the TF antigen can be identified either by the beta-glycosidic or protein backbone connection of the carbohydrate structure.
Potential targets for both passive and active cancer immunotherapies include carbohydrate tumor antigens on glycolipids and glycoproteins. These extremely ample antigens are de novo up-regulated due to variations in the composite glycosylation apparatus of tumor cells, which involves various groups of enzymes such as epimerases, glucosidases, glycosyltransferases, and nucleotide sugar transporters. Several proteins or lipid-bound carbohydrate tumor antigens are usually pronounced, for instance, GD2, GD3, GM2, Sialyl-Lea, Globo H, LeY, fucosylated GMI, and the mucin core structures including TF, Tn, and Sialyl-Tn (Kurtenkov et al., 2018). The most abundant antigens are the carbohydrate tumor, as compared to protein tumor antigens, making them the appropriate targets specifically for antibodies. A good example is the high conveyed protein tumor markers such as Her-2/neu and TF, which show about 106 and 107 copies per cell respectively (Kurtenkov et al., 2018). In addition, just like carbohydrate structures, cellular immune responses are also potential targets for humoral. Among the protein and carbohydrate tumor-based antigens, Thomsen-Friedenreich is an oncofetal antigen that shows an exceptional tumor specificity appearing nearly in all tumors.
The changed glycosylation frequently identified in cancer cells leads to the manifestation of changed glycopeptide epitopes, and tumor-associated glycans (TAG) which are in most cases autoimmunogenic and are usually recognized by autoantibodies. A variety of adaptive anti-glycan and natural Abs occur in human serum in health and disease, showcasing a fairly stable level through a distinct duration in healthy people. The majority of these Abs are brought about by the adaptive and innate immune reaction to microbial carbohydrates (Kurtenkov et al., 2018). Thomsen-Friedenreich is an immunoreactive glycoantigen, CD176 (Galβ1-3GalNAcα-O-Ser/Thr (Core 1) structure) which encompasses about ninety percent of human carcinomas, but it is hardly expressed in healthy tissues. TF-specific Abs which occur naturally are expressed at reduced levels in cancer, and they are linked with tumor progression as well as patient survival, signifying the significant function of anti-TF Abs in tumor immunosurveillance (Kurtenkov et al., 2018). Both humanized mAbs and murine to TF exhibited in vivo and in vitro action towards TF-positive human breast cancer was also depicted in a xenograft model in SCID mice.
Immunoglobulins (Igs) are glycosylated molecules that are influenced strongly by N-glycans of the Fc-fragment. Others affected include the Fc-mediated effector mechanisms and the IgG-Fcγ receptor interactions (Kurtenkov et al., 2018). Agalactosylated IgGs have been proved to show an amplified inflammatory activity, while sialylated Abs exhibit an anti-inflammatory effect. In comparison to healthy people, serum IgG glycosylation shows a noticeable change in people with infections, autoimmune diseases, and tumors, including those with breast cancer. The profiling of this serum shows a prognostic and diagnostic potential in various malignancies and breasts. However, of importance to note is that the total serum IgG glycosylation may considerably vary from that of antigen-specific, a case which signifies the existence of disease-specific IgG variations of possible clinical importance (Kurtenkov et al., 2018). Due to the significant role of glycans in the Abs’ functional behavior, and the possible construction of Ab glycoforms, the Abs’ glycodiversity has become a topic of interest.
Even though antibodies are proved to be heterogeneous by functionality, there is no clear data on the glycodiversity of Abs on antigens linked to tumors. This includes the tumor-associated glycans (TAG), and cancer biomarkers that are currently in use. The modified glycosylatin which is identified in cancer cells can lead to the expression of changed glycopeptide epitopes, and TAGs that are autoimmunogenic, and are recognized by autoantibodies. The immunoreactive TF glycoantigen is shown in about 90% of human carcinomas and it is never expressed in healthy tissues. TF-specific Abs which occur naturally are at decreased levels in cancer and the same is associated with the progression of tumor and patient survival. This shows the main function of anti-TF Abs immunosurveillance of tumors (Sindrewicz et al., 2016). Thus, TF glycotope occurring naturally show cancer-specific changes identified in the early phases of breast cancer. Unsubstituted and unmasked TF transpires in approximately ninety percent of all cancers. Hence, there occurs a direct correlation between tumor progression and the manifestation of TF in varied forms of cancer.
Synthesis of TF Antigen
The interaction of the Thomsen-Friedenreich-antigen with galectin 1 and 3 gives rise to tumor cell accumulation and enhances cancer metastasis, as well as T-cell apoptosis in the epithelial nerve. With the purpose of exhausting the exploration of multivalent binding between the galectin-3 and TF-antigen, the last is enzymatically produced in large quantities through the use of GalNAc (α1-EG3-azide as the acceptor substrate, and glycosynthase BgaC/Glu233Gly is utilized in the reaction. Consequently, alkynyl-functionalized bovine serum albumin is included through a copper (I)-catalyzed alkyne-azide cycloaddition. Neo-glycoproteins containing tunable glycan multivalency are yielded by the procedure (Hoffmann et al., 2020). At the same time, glycan residues and densities of 53 and 2 per protein molecule are respectively obtained by use of a regulated alkynyl modification. The number of glycans is then measured by trinitrobenzene sulfonic acid assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Hoffmann et al., 2020). The resulting binding efficacy of the human galectin-3with neo-glycoproteins and the effect of multivalency is then examined and evaluated by the use of an enzyme-linked lectin assay.
Retrained neo-glycoproteins of various concentrations show an increasing glycan density with binding of Gal-3. Yet, the presentation of multivalent glycan shows no high binding affinity (Hoffmann et al., 2020). Preventing Gal-3 binding to asialofetuin is normally active, and the relative strength of inhibition can be raised by a factor of 142 for neo-glycoproteins, which display 10 glycans/protein (Hoffmann et al., 2020). This condition contrasts the high decorated inhibitors which only show a 2-fold increase. Thomsen-Friedenreich-antigen is a carbohydrate structure that is of great significance in the O-glycosylation of glycoproteins of mammals. Originating from its antecedent GalNAc (α-O-Ser/Thr (Tn-antigen), it acts as a support for multifaceted mucin-type O-glycan and longer structures. There are usually increased levels of TF-and Tn- antigen which happen in 70–90% of all human carcinomas (Hoffmann et al., 2020). These are typically found on the glycoprotein mucin-1 (MUC-1) which binds the cell surface and are said to act as pancarcinoma antigens.
The TF-antigen is a powerful ligand in both galectin-3 (Gal-3) and galectin-1 (Gal-1) arbitrated cell interactions, with the former showing a stronger binding effect. Gal-3 is conveyed on the outside sheath of carcinoma and endothelial cells, it muddles to the TF-antigen shown on MUC-1 in both flow and static conditions. Circulating Gal-3 shows that it binds to MUC-1 on the superficial layer of cancer cells, thus separating the surface of the cell, such that the adhesion molecules are clearly exposed leading to the formation of tumor and aggregation. Therefore, it is important for TF-antigen to interact with Gal-3 because it aids in a homotypic combination of cancer cells, and in the original propagation and adhesion of carcinoma cells (Hoffmann et al., 2020). Owing to the fact that these mechanisms form the initial steps of cancer proliferation and metastasis, tiny molecule antagonists which have the possibility of inhibiting the binding of Gal-3 are being developed.
Moreover, multivalent synthetic ligands are also in the final stages of being confirmed as possible folders of Gal-3 with KD-values. Particularly, multivalent neo-glycoproteins (NGPs) based on bovine serum albumin (BSA) which displays thiodigalactosides, GalNAc (β1–4) GlcNAc (β1–3) Gal (β-GlcNAc or LacdiNAc-LacNAc motif, are also confirmed as actual ligands for Gal-3 with sub-nanomolar KD-values (Hoffmann et al., 2020). Presenting one TF-antigen ligand on synthetic glycopeptides is good since it allows it to acquire an affinity for Gal-3 binding, in comparison to non-conjugated glycomimetics and TF-antigen. On the other hand, presenting a multivalent TF-antigen activates the Gal-3 binding as confirmed by the antigen showing an anti-freeze protein from codfish, a case that overpowers T-cell apoptosis as well as prostate cancer metastasis in mice (Hoffmann et al., 2020). BSA-based NGPs, the tunable and the synthesis of multivalent demonstration of Gal (β1, 3) GalNAc (α1-EG3-azide (TF-antigen-azide) shows a considerable binding interaction with Gal-3.
Comparing TF Antigen to Normal Cells
In terms of growth, normal cells usually stop reproducing/growing when there are enough cells. For instance, if cells are produced to help in repairing skin cuts, when they are sufficient to seal the hole, more are no longer produced. In contrast, TF antigen (epitope Galbeta1-3GalNAcalpha-O-) is referred to as a carcinoma-associated antigen for a long time now. In normal tissue, TF is covered by glycosyl structures, thus creating the glycoproteins identified to account for the MN-blood group, but the same molecules are uncovered in malignant tissue. The occurrence of this antigen in normal tissue is limited to some immunologically advantaged areas. The antigen does not stop growing even when the cells are enough, and the continued growth leads to malignancy and tumor formation. In the body, each gene has a blueprint coded with a different protein (Hutchins, 2017). These proteins are growth factors, and they initiate the growth and division of cells.
In case the gene that codes a particular protein is stuck in a position due to mutation (the case of TF antigen), the factors for growth continue to be produced, and the cells continue growing. One of the common post-translational alterations of the proteins of the cell membrane is glycosylation. The N-linked type of glycosylation is characterized by carbohydrates attached to the asparagine residues amide group of proteins in the sequence Asn–X–Ser/Thr (Hutchins, 2017). In contrast, the classical N-acetyl-galactosamine, O-linked mucin-type glycosylation is covalently attached to the hydroxyl group of threonine residue or serine of the backbone protein. The O-linked protein glycosylation takes place in the course of protein movement via ER–Golgi pathway by progressive absorption of monosaccharides. This process is normally controlled by various factors, including glycoprotein substrates and relative abundances of glycosyltransferases as well as, sugar-donor molecule availability (Hutchins, 2017). Unlike normal cells which interact with others and respond to signals from nearby cells, tumor-associated antigen does not react to any signal to stop growing. Therefore, the changed glycosylation of cell membrane proteins acts as a common feature of oncogenesis cancer progression.
This glycosylation may typically change to include the occurrence of truncated and incomplete glycan structures, accumulated glycan precursors, or the development of novel carbohydrate epitopes which are tumor-specific. While normal cells either die (undergo apoptosis) or are revamped whenever they get damaged or get old, tumor-associated cells undergo metastasis. For example, a healthy cell has a protein called p53 whose role is to check whether a certain cell is too damaged to repair, a case in which it advises the cell to kill itself. In case p53 is inactive or aberrant, which may be as a result of a mutation in its gene, then damaged and old cells may be allowed to reproduce (Hutchins, 2017). A known fact is that the TF-antigen is involved in the adhesion of tumor cells to the endothelium, through a mechanism involving the recruitment of Galectin-3 and MUC-1, forming the first step in metastasis. However, the pathway might be blocked by increasing molecules, thereby giving way to therapeutic intervention. Whenever a normal cell becomes damaged, it is identified by the immune system through the lymphocytes and it is then removed.
Normal cells do not metastasize but stay and are located in the area of the body where they are supposed to belong, because they have adhesion molecules that result in stickiness. TF-antigen may lack these molecules hence they detach and travel through the lymphatic system and the bloodstream to other organs of the body. Their growth then continues in these other areas forming tumors distinct from the original one (Hutchins, 2017). The glycosylation changes in TF- antigen on cancer cell surface virtually take part in regulating cell social behaviors and have been gradually recognized to play a vital role in cancer progression, development, and metastasis. Amplified frequency of short carbohydrate structure Galβ1–3GalNAcα known as oncofetal Thomsen–Friedenreich (TF or T) antigen, is among the most predominant glycosylation modifications in human carcinomas. The appearance of normal cells and that of TF-antigen is quite different, especially under a microscope. The latter exhibit variability in size, some are either smaller or larger than normal (Hutchins, 2017). Tumor-based cells have an aberrant shape, and their nucleus is usually darker and larger than that of normal cells.
The excess DNA contained by the antigen is the reason for the dark nucleus. They also have an unusual number of chromosomes with a disorganized fashion in their arrangement. Thomsen-Friedenreich is a precursor molecule of the MN-blood group antigens, but in normal tissue, the same is coated by glycosyl structures. While normal cells have the maturity, tumor-associated cells do not since they divide and grow rapidly before they mature, and thus remain undifferentiated. The TF-antigen is the fundamental structure of O-linked mucin-type glycans (Hutchins, 2017). Therefore, in normal epithelium, the structure is covered with deposits of sugar which form composite and branched glycans amended by fucosylation, sialidation, or sulfation. Low levels of TF expression are infrequently reported in normal epithelium, a case alleged to be linked to the use of different explicit analytic tools.
In terms of energy consumption, in comparison to normal cells, tumor-associated cells use more energy for proliferation, which is by exhaustion of high amounts of glucose, through transportation of receptors. The overexpressed sugar-binding which is located in the cellular membrane also consumes a lot of energy. In addition, the tumor-based cells can also evade the immune system for quite some time, enough to develop into a tumor either by secreting chemicals that can make immune cells inactive or by escaping detection. Whereas the TF antigen may at times not be functional, normal cells always function the way they are supposed to. Angiogenesis is the process of the cells that involves the attraction of blood vessels to grow and feed the tissue, a process undergone by normal cells as part of their growth (Hutchins, 2017). On the other hand, tumor-based cells undertake this process even when they do not need to grow.
Aptamers and Selex as Biomarkers for TACAs
The precision of cancer medicine entirely relies on the characterization of molecules associated with tumors, which is generally the genetic material, to give guidance to the therapy of an individual patient. However, unusual variations in the human genome, which are increasingly revealed by the next generation developing rapidly, and the sequencing technologies are some of the factors of molecular changes that determine cancer and pre-cancer progress. Proteins are known to control cellular activity, but besides gene transcription, their functions and dysregulations in the post-translational modification (PTM) machinery are improved by a surfeit of posttranslational modifications. This leads to the achievement of abnormal protein functions used in tumorigenesis (Díaz-Fernández et al., 2020). There are different types of PTMs, and among them, glycosylation is predominantly vital because it plays critical roles in various cellular processes involved in the progression of cancer, including tumor immune surveillance and cell differentiation, signaling, and cell-matrix interaction, invasion and metastasis formation.
In spite of the unusual glycosylation patterns in some proteins such as a prostate-specific antigen, α-fetoprotein, or mucins, which have been defined as an essential characteristic of malevolent changes, the glycoproteins level is monitored. Glycan moiety is basically ignored, and it is not scrutinized in clinically cancer prognosis and diagnosis. Another informative but perplexing approach is the discovery of explicit glycoforms of the protein biomarkers. Basically, there are two strategies that aid in attaining information on the levels of a protein linked to a particular glycan (Díaz-Fernández et al., 2020). The first method is founded on mass spectrometry in conjunction with various methods of separation, following enzymatic digestion of the sample to be used. This approach is an influential tool for glycopeptide mapping, though there exist significant practical obstacles. These include the crucial matrix influences in biological fluids, and the poor ionization efficacy of glycopeptides, which gives rise to a limited sensitivity.
The other method uses a receptor known to be select the protein, and a good instance is an aptamer or an antibody, and a lectin. These have the capability of recognizing explicit carbohydrate structures, even though the glycosylation of antibodies makes the analysis in the common immune-lectin assays complex (Díaz-Fernández et al., 2020). There is also a possibility of detecting protein-specific glycosylation by artificially connecting a protein receptor, aptamer, or an antibody with a glycan that is engineered metabolically, mostly with azide-containing sugars. But a major drawback is the danger of defective links (open bridges) commonly used as a basis for false-positive results.
An aptamer is a good alternative to antibodies as explicit receptors, and there have been various efforts to help in guiding their choice toward sugar moiety. This is done by either using the whole glycoprotein or glycosylated-peptide fragments as targets (Díaz-Fernández et al., 2020). These approaches are helped by the inherent capacity of boronic acid to react with diols in the sugar moiety, by integrating the group in the sequence of the aptamer or in supporting the protein immobilization. Currently, the aptamers obtained so far show an ability to either recognize a specific carbohydrate epitope, or different glycan structures irrespective of the protein where it is attached. Just one aptamer has been claimed to have the ability to recognize both glycan structure and peptide, but it only identifies short peptides rather than the complete protein (Díaz-Fernández et al., 2020). Despite its predicted capability in cancer prognostic and diagnostic, no synthetic receptor with the competence of integrating the recognition of both the peptide and the glycan moiety surrounds an intact glycoprotein.
Though the peptide and the glycan moiety which surrounds an intact glycoprotein can be diagnosed by the aptamer, a recent study has proved that a mammalian lectin, Dectin-1, identifies a glycan/peptide part in IgG antibodies. A striking target for the model system is the human prostate-specific antigen (HPSA) since it is a glycoprotein (Díaz-Fernández et al., 2020). Its level of serum is used as a typical assessment for diagnosing, monitoring, and screening the progress of the disease in prostate cancer. The test cannot however differentiate aggressive from indolent disease, and it does not have enough diagnostic selectivity. If the ailment is absent, HPSA contains just one N-glycosylation spot at asparagine-61 (Asn61 = Asn69 according to another assignment), with a unanimity biantennary glycan structure which represents ∼80% of the total glycoforms identified (Díaz-Fernández et al., 2020). Various changes in the oligosaccharides profile are linked to the development of prostate cancer (Díaz-Fernández et al., 2020). Essentially, a straightforward method that can help advance the usage of HPSA as a cancer biomarker is to acquire aptamers that have the ability to recognize glycosylation variations. In any case, these differences may arise at the specific site in the protein.
In the world, gastric cancer is among the leading causes of death related to cancer. However, this type of cancer has remained difficult to cure because there is no early biomarkers detection, and it is diagnosed at the advanced stages in many patients (Pan et al., 2018). Over the last decades, clinical management has been using tumor markers or detection of biomarkers to help in the identification prediction of prognosis, screening, and reappearance, as well as monitoring after treatment. The most frequently used biomarkers for gastrointestinal cancer are classified as tumor-associated antigens, including cancer antigen 19–9 (CA19-9), 50 (CA50), and 72–4 (CA72-4), and carcinoembryonic antigen (CEA). The latter has a molecular weight of 180–200 kD, and it is a glycoprotein on the cell surface that has a vital function in intracellular signaling and cell association.
Antigen 50 (CA50) has a molecular weight of ~210 kD and has been demarcated by monoclonal antibody C 50 established alongside colorectal cancer (CRC) cell line COLO-205. It contains increased levels of serum and can be detected in a range of menaces, particularly gastrointestinal cancers. Cancer antigen 72–4 (CA72-4) has a weight of 220–400 kD, it is a mucin-like tumor with a high molecular weight. It is often contemplated to be the leading tumor marker for gastric carcinoma as a result of its superior sensitivity. Presently, radio-immunoassay (RIA) and immunoassay (EIA) enzymes are regularly used to detect tumor markers. However, one area of consideration is the fact that the interactions between an antigen and an antibody may be highly specific (Pan et al., 2018). For clinical applications, standardization and harmonization of the assays are drawbacks and challenges of immunoassays. The reason is that diverse productions of the commercial detection kits of tumor markers might make dissimilar epitopes for the immunoassays. This, in turn, culminates in variations in quality and challenges related to standardization.
Disparities in inducing the immune response in the biological system may equally play a part in the ambiguity of the quality, with the production likely to be affected by lot-to-lot variation. A marginal disconcertion of conformational epitopes on natural proteins might result in a failure in penetration against the corresponding antigen by a monoclonal antibody (Pan et al., 2018). The short shelf life and the moderately low stability of immunoassays, as well as their sensitivity cause challenge for recurrent standardizations and vacillations of the results. The encounters trigger a need for developing a standardized approach for tumor detection markers, more specifically gastric cancer, for clinical applications. RNA and DNA oligonucleotides aptamers have the high selectivity and affinity to bind various targets (Pan et al., 2018). The approach employed for screening explicit aptamers is referred to as Systematic Evolution of Ligands by Exponential enrichment (SELEX), and it was established in 1990. It is an in vitro selection method which permits a concurrent screening of nucleic acid molecules up to 1015 for each target selected.
Though it is possible to select the target to be optimized and approved under a given condition, aptamers are applied in both diagnosis and therapy. This includes Pegaptanib, an FDA accepted anti-vascular endothelial factor for growth (anti-VEGF). The growing interest in aptamers is attributed to their superior properties as well as their versatility. In general, they are more stable than antibodies and their modification significantly lengthens their shelf life and enhances their functionality. In terms of reproducibility and dependability, the quality of aptamers is consistent due to their purification and synthesis which are robust under stings and can be controlled by a machine. Further, the optimization and selection of various aptamers for production on a large scale are better and simple as compared to monoclonal antibodies. Therefore, aptamer selection and the consequent application is relatively auspicious in medical services and biological research (Pan et al., 2018). The progress of SELEX has been rapid over the last decade, making the aptamer development for clinical and commercial use more interesting and efficient.
SELEX is presently integrated effectively in microfluidic chip-based systems to enable it to support the screening aptamers alongside the protein targets having higher affinities. Besides, cell SELEX sightsees the cell surface epitopes manifestation and differentiates between diverse types of target cells. There is a growing number of DNA/RNA aptamers which target gastrointestinal cancer cells and biomarkers being discovered in recent years (Pan et al., 2018). For instance, RNA aptamer YJ-1 tangles explicitly to CEA-positive cells and repressed homotypic invasion, aggregation, and migration and by CEA-positive cells in mice (Pan et al., 2018). DNA aptamers attached to CEA’s lgV-like N domain have been proved to impede cell adhesion qualities of cancer cells. In addition, the aptamer cy-apt20 which detects DNA targets human gastrointestinal carcinoma AGS cells, and it displayed nominal identification to typical gastric epithelial GES-1 cells. Other aptamers which have been identified so far include cancer stem cells (CR-CSCs)/CRC-specific. Among these, some have shown considerable attractions to the particular target cells. Important noted is that aptamers such as CA72-4 imagined to target other gastrointestinal cancer biomarkers are yet to be reported.
Conclusion
As discussed above, the abundant and complex biomolecules which play an essential role in various cellular interactions are carbohydrates. The functions include signaling other cell surface receptors or cellular molecules. A variety of oligosaccharide and monosaccharide residues are linked by glycosidic connections to form important glycoconjugates, which include glycolipids, glycoproteins, and glycosylated natural products. Additionally, the biosynthesis of these glycans is usually regulated by various enzymes, and any aberration in the structure of the cell surface allows them to encrypt information that is needed for disease progression. Thus, carbohydrates have the ability to induce glycan-mediated interactions and are normally embattled as pharmaceutical therapeutic agents with an aim of treating different pathological diseases.
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