1. Introduction:

1.1 Background of the study:

Cancer is one of the major public health problems across the globe. The reports of American Cancer Society have revealed that cancer has been considered as the major burden among the developed and developing countries (DeSantis et al., 2015). It has been estimated that almost 14.1 million new cancer cases with occurrence of approximately 8.2 million deaths during 2012 across the world. Hence, this global scenario of the impact of cancer across the world has evolved the progression in traditional methods of cancer therapy involving chemotherapy, radiations, and surgery since the last few years. Despite all these efforts in traditional methods of therapy towards cancer, it has still been difficult to cure and prevent it. Hence, imperative methods have been developed for combating the serious threats of cancer. Therefore, vaccines and immunotherapy against cancer have been developed for the prevention and treatment of cancer.  The therapeutic cancer vaccines have been always considered an important ailment towards the eradication of the initial tumors and blocks the activity of the malignant cells from converting into tumor cells. Hence, these vaccines have been effective in boosting the immune response against the tumor cells (Chuang et al., 2013; Heimburg-Molinaro et al., 2011; Hevey& Ling, 2012).

Tumor-associated carbohydrate antigens (TACAs) is one of the major forms of product present in the form of carbohydrate-based antigen which is clearly defined by the monoclonal antibodies. Tumor-associated carbohydrate antigens (TACAs) are known as the type of molecular markers that are more frequently overexpressed on the human tumor cells, helps in distinguishing the tumor cells from the normal cells (Bergquist et al., 2016; Aldakkak et al., 2015; Bauer et al., 2013). The presence of the types of carbohydrate structures, also called the glycan structure helps in this distinction. The alterations in the protein glycosylation and expression that has occurred due to variations in the glycan structures that further correlates with the development and progression of human diseases and cancer. The glycans are responsible for regulating the various aspects of tumor development involving invasion, proliferation, and metastasis. This has led towards various changes in the glycosylation patterns which have been detected in the array of human cancers. TACAs are always prioritized towards the development of novel carbohydrate-based anti-cancer vaccines. Hence, the recent years researchhas shown progression towards the development of synthetic, carbohydrate-based antitumor vaccines for improving the immune responses that are associated with targeting of the specific antigens. Carbohydrates are complex biomolecules that play important roles in various cellular interactions involving signaling towards other cellular molecules and cell surface receptors (Sartorius et al., 2016).  Hence, carbohydrates are now considered effective for the establishment for clinically relevant antigens along with other cancer vaccines which are developed based on carbohydrates.

The carbohydrate antigens are usually considered to be T-cell independent while producing a weaker immune response (Payandeh et al., 2018). This is the reason why the use of carbohydrate antigens has been referred to as a challenging approach towards the use of carbohydrate antigens in cancer vaccines evolving various difficulties associated with overcoming immunotolerant and immunosuppression. Carbohydrate antigens do not possess inherent immunogenicity with bacterial antigens and related techniques towards the improvement of immunological recognition. This includes the involvement of TACA that are the form of chemically modified antigens that increases the immunological reactivity (Hutchins et al., 2017; Qin et al., 2014). The evolution of TACAs has been known to be time consuming and its separation from the tumor cells have been found to be difficult. Hence, various strategies involving oligosaccharide synthetic strategies with chemo-synthetic and chemo-enzymatic methods have been developed for obtaining pure and homogenous TACAs at larger scale (Yin et al., 2017; Zhou et al., 2017). Hence, these technological advances have enabled the development of TACA-based cancer vaccines. The most known technique for coupling TACA to the carrier proteins for producing semi-synthetic glycoproteins.

 

1.2 Literature review

1.2.1 Tumor associated carbohydrate antigens (TACAs)

Cancer is a main source of death around the world. The worldwide cancer trouble, which has been assessed as 18.1 million new cases and 9.6 million passings in 2018, is anticipated to ascend to 29.5 million new rates in 2040 (WHO, 2018). The disturbing expansion in in treatment of cancer emphasizes the earnest need to create innovative methodologies for more powerful and less harmful disease medicines supplementing ordinary therapeutics like chemotherapy, radiotherapy, medical procedure, and palliative consideration. Disease immunotherapy holds extraordinary guarantees in cancer treatment, which was named as the Breakthrough of the Year in 2013 by Science (Couzin-Frankel, 2013).

Tumor related carbohydrate antigens (TACAs) are a class of appealing antigens for the advancement of anticancer immunotherapy. Other than monoclonal antibodies and immunizations, chimeric antigen receptor (CAR), also known as fanciful immunoreceptors or T cells receptors and bispecific antibodies (BsAbs) have been used in the development of TACA that are further effective in treating cancer by evolving resistant framework towards treatment of cancer. Two TACAs involving the GD2 ganglioside, and the mucin-1 (MUC1) protein have been found to be effective in combating the battle of cancer among the cancer patients. The most recent advances in chimeric antigen receptor (CAR), T cell receptors and bispecific antibodies have been introduced that focuses on the antigens such as GD2 ganglioside and mucin-1 (MUC1) protein. The parts of co-stimulatory atoms, designs of the groupings for antigen restricting, techniques for CAR and immunizer development have been proved to be effective towards upgradation of strong tumor entrance and lessen T cell depletion (Rashidi Jehanabad, and Huang, 2020).

Various results have been presented for treatment of cancer. The chimeric antigen receptors (CAR T cells) and BsAbs that focusses on GD2 and MUC1 have shown effective results in successful treatment of strong tumors (Hakomori, 2001). Major focus have been provided towards improvement of anticancer antibodies for development of TACAs having using innovative strategies for developing antigen structures and support the transporter moieties to convey the antigens to the safe framework so that the resistant actuation components including adjuvants and cytotoxic T cell epitopes can fuse effectively during the development of immunization (Wei et al., 2018). Other than immunizations, chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs) have been considered as engaging immunotherapeutic systems in treating canceras per the confirmation by the FDA (Riley et al., 2019). Major advancement has been successfully implementedfor improvement of CAR T cells and BsAbs that majorly focusses on two TACAs including gangliosides GD2 and glycoprotein mucin-1 (MUC1).

One of the reports of advancement therapies revealed that the normal danger to a lady in the Western world creating breast cancer (Bca) eventually in their life is roughly 12% (Sun et al., 2018). TACAs have been proved to be effective in improving Bca cell endurance and the ganglioside GD2 has been used in conjugation with TACA antibodies to restrain breast cancer. Other studies have revealed that insusceptibility to Lewis y and GD2 could be initiated through the creation of a carb copying peptide (CMP) that imitates the two receptors. The CMP (P10s) in Phase I portion heightening preliminaries with the adjuvant MONTANIDE ISA 51 VG that was acted in Bca patients over a multi week time frame in which neutralizer reactions to P10s, GD2, and LeY happened overall six of the patients who were evaluated. Vaccination utilizing the p10 immunization was very much endured and initiated useful in generation of antibodies thatrecommends its clinical advantages (Hutchins et al., 2017).

Another method including a multiplex dot-based estimation of the humoral insusceptible reactions against tumor-related antigens has been used to survey their systems of activity. In the investigation, they represented the advantages of using P10s starch-based immunization for enacting Natural Killer (NK) cells that helps in tumor invasion, and also shapes the versatile reactions towards a T partner type 1 (Th1) profile in mouse models. The results also showed that stage IV metastatic Bca patients had a Th1 cytokine climate set apart by significant IFN-c height (Abbas et al., 2018). P10s-PADRE inoculation was appeared to actuate the outflow of supportive apoptotic antibodies that were caspase-3 ward prompting ADCC focusing on human Bca cells in vitro, further stressing the job of initiating NK cells.

The inoculation with the CMP immunization mediates with tumor NK reactions. This antibody methodology is presently in human clinical preliminaries for additional appraisal (Trial number: NCT01390064). Moreover, an enormous, randomized stage II/III preliminary was performed with Globo H-KLH form antibody (OPT-822) in 349 metastatic breast cancer patients who had\ 2 scenes of reformist sickness, and who achieved stable illness after in excess of a course of treatment (Huang et al. 2016). Patients were randomized 2:1 to get either the antibody or PBS control, alongside a low portion of cyclophosphamide. Movement Free endurance and generally endurance were essentially expanded in half of patients who grew early explicit immunizer reactions to OPT-822 regardless of the breast cancer subtype.

 

1.2.2 Mechanism of TACAs

Carbohydrates (glycans), just as glycoproteins and glycolipids, are significant cell surface segments, and numerous monoclonal antibodies (mAbs) have been created to focus on these particles for different applications. Changes in cell glycosylation are normal in cancer because of atypical articulation of glycosyltransferases, glycosidases, and carriers, just as contrasts in the plenitude of Carbohydrates building blocks (Ben-Arye, et al., 2017). Such adjustments can bring about remarkable antigenic glycans alluded as Tumor-Associated Carbohydrate Antigens (TACAs). TACAs are utilized clinically as illness markers, for instance, sialyl Lewis a (sLea ) is utilized for arranging of pancreatic disease. Furthermore, focusing on TACA epitopes has gotten huge for overseeing different human tumors (Amon, et al., 2018). A counter acting agent against the ganglioside GD2 has shown valuable impacts in neuroblastoma treatment and a few other antibodies of Carbohydrates antibodies are at present in clinical preliminaries. AlthoughmAbs are clinically significant devices, antibodies against starches will in general have low proclivity and intricate or blended explicitness. What’s more, the acknowledgment of the glycan antigen can rely upon glycan thickness, valency, introduction, and adaptability (Thieker, et al., 2016). The lack of high liking as well as high explicitness antibodies against Carbohydrates targets is a vital limit in abusing glycans as illness markers or helpful targets.

Antibodies receptive with TACAs are proposed to be particular for bunched TACA even within the sight of typical tissue. Thus, TACAs coming about either from limited conveyance on typical cell surface, fragmented union or neosynthesis amass in high thickness or bunched design (potentially in novel conformities comparative with glycans communicated on ordinary tissue) at the tumor cell surface specifically brings about an absence of insusceptible interceded tissue harm notwithstanding that antibodies can tie to “ordinary” tissue (Song et al., 2017). Meddling with flagging components related with TACA articulation can stop the development of tumors and forestall the advancement of metastases. TACA-responsive antibodies are referred to as proapoptotic both as monoclonal antibodies and as a component of insusceptible reconnaissance. Both the LeY and ganglioside GD2 antigens can control flagging cycles in a few distinct manners that loans themselves to the movement of cancer (Orsi et al., 2017). Exploiting the polyspecific idea of antibodies, we created P10s as a Pan-immunogen, responding with numerous antibodies of TACA antibodies and lectins. Consequently, the acceptance of polyreactive, proapoptotic antibodies would be a gainful component in cancer immunotherapy draws near. The acceptance of such antibodies can accommodate longer-term endurance of cancer subjects, as such antibodies are clinically corresponded with long haul endurance (Kieber-Emmons et al., 2017).

 

1.2.3 Use of TACA in potential cancer vaccine 

The synthesis of TACA analogs is an integral asset to address the characteristic restrictions of these inescapable tumor markers. The Tn antigen is a TACA of incredible interest in cancer vaccine growth, since it is primarily basic, comprehensively communicated in numerous adenocarcinomas, however concealed or practically missing on typical cells (Bermejo et al., 2020).  Bermejo et al. (2020) sums up the manufactured analogs detailed as a potential system to overhaul the poor immunogenic local Tn. The benefits of the engineered concerning the local antigen, and their utilization (assuming any) in immunomodulating developments have likewise been talked about. Extraordinary consideration has been devoted to basically compelled Tn analogs, which all the more viable emulate the introduction of the local antigen and its acknowledgment by Tn antibodies (Fernández et al., 2016).

In the previous century, the examination on the immunotherapy of disease has shown energizing clinical outcomes, alongside some critical impediments, which influence an expansive utilization of cancer growth immunotherapy in facilities (Force et al., 2019). A basic angle meriting consideration is the helpless insusceptible reaction typically inspired against disease cells, which is likely because of the immunosuppressive instruments physiologically applied to forestall an unnecessary and hurtful resistant reaction (Dine et al., 2017). Albeit critical advances have been accomplished in TACA-based cancer treatment, no immunization has as of now been endorsed, principally because evoking defensive, T-cell intervened, resistant reaction against saccharides antigens has been extremely testing modern. In this unique situation, it has been shown as of late that inhibitory flagging pathways or invulnerable designated spots forestall the mounting of an insusceptible reaction (Huck et al., 2018).

Antibodies focusing on designated spots have shown adequacy in cancer treatment and some are as of now available. In any case, not everything malignancies can be effectively treated with designated spot inhibitors and resistant unfavorable impacts have been regularly enlisted. All the gathered proof plainly shows that the quest for malignancy medicines to expand quiet advantages and to decrease antagonistic results is as yet a difficult undertaking, yet additionally recommends that blend treatments are probably going to be the eventual fate of immuno oncology. Cancer vaccines, TACA-based antibodies specifically, along with designated spot inhibitors, could be helpfully consolidated to improve insusceptible reactions in tumor-bearing hosts, tending to the fundamental issues of immunotherapy. Surely, two stage I clinical preliminaries are at present being worked on testing neoantigen immunizations in combination with obstructing antibodies to CTLA-4 and PD-1 (Gibney et al., 2015). In this problem area, the decision of antigen mimetic for malignancy antibody advancement could address an effective system to break the known resistant resilience versus TACAs and to conquer the in vivo shakiness of saccharides antigens to glycosidases.

Despite the extraordinary capability of TACAs-based antitumor antibodies as a profoundly powerful and explicit malignancy immunotherapy, they are as yet dealing with major issues. Right off the bat, accessibility of adequate TACAs. The carb antigens applied to malignancy antibodies were segregated from tumor cells at first, and the heterogeneity of the sugar chains on the cell surface makes the isolating activity arduous and tedious (Feng et al., 2016). Be that as it may, this issue has been settled fundamentally with the roaring improvement of glycobiology and oligosaccharide amalgamation as of late. Strong stage amalgamation of oligosaccharides, one-pot blend contingent upon the action of glycosyl benefactors, initiation procedure autonomous of movement of glycosyl contributors, chemo-enzymatic union of oligosaccharides and a progression of oligosaccharide union advancement give new ways to the union of plentiful, high immaculateness and fundamentally uniform TACAs. Also, most TACAs are T-cell-autonomous antigens. They don’t tie with the significant histocompatibility buildings (MHCs) straightforwardly and can’t initiate T-cells without anyone else. Rather than peptides and proteins, sugar antigens neglect to actuate T-cell-intervened resistance which is basic for cancer immunotherapy (Bray et al., 2018).

TACAs alone can just pitifully actuate the B-cells. Without extra assistance from aide T-cells (Th-cells), low titers of the low-partiality IgM antibodies are created. To produce IgG by B-cells, two signs are required: an antigen-explicit sign conveyed by means of cross-connecting of B-cell receptors (BCRs) on the phone surface and a co-stimulatory cytokine signal conveyed by Th-cells. To change over the invulnerability type to IgG-type, cells need to cooperate with Th-cells. It is realized that peptides are the standard epitopes to bring out the T-cells reaction through restricting with MHCs. Subsequently, an old-style plan of sugar-based immunizations is to form TACAs with a protein or peptide transporter containing set up T-cell epitopes (Balmaña et al., 2015).

Antigen-presenting cells (APCs) will catch and disguise the form antibodies when they enter the human body and divide them into peptides. At that point, the peptide epitopes onto the cell surface as buildings with MHC class II (MHC II) particles (Llorens et al., 2015). These edifices present antigens to T-cells and they are vital for T lymphocyte enactment. The enacted T-cells will deliver cytokines and Th-cells will advance the B-cell expansion and produce IgG antibodies and separate to memory B lymphocytes which can endure a more extended period. Memory B lymphocytes can react quickly and produce high fondness IgG antibodies when similar antigens present (Biswas et al., 2015).

 

 

2. Aims and Objectives

The primary objective of this project is to determine whether the tumor associated carbohydrate antigen would function as a potential vaccine and induce meaningful immune responses in human and murine macrophages.

 

2.1.1 Hypothesis

It was hypothesised that Zwitterionic Polysaccharide capsular proteinwill induce strong immune responses in macrophages and boost innate responses towards cancer antigens. This hypothesis will be directly tested by comparative analysis of the response of macrophages towards cancer tumor associated carbohydrate antigen along with formulation with Zwitterionic Polysaccharide capsular protein. This results into analyzing the correlation with increased production of T-cell-dependent immune responses and antigen processing. This study will adopt the concept of formulating a Zwitterionic Polysaccharide capsular protein needed for preparing off-shelf vaccines to do in vitro in vivo tests. The tests will use Zwitterionic Polysaccharide capsular protein and membrane antigens derived from tumor cell for inducing the immune activation of both human and murine macrophages.

2.1.2 Specific Aims

• Determine whether an increase in the production of proinflammatory cytokines by macrophages is observed following the exposure of macrophages to Zwitterionic Polysaccharide capsular protein. Objective: To quantify cytokines produced by macrophages by analysing growth media using ELISA and by analysing gene expression in macrophages using real-time PCR
• Determine whether zwitterionic polysaccharides treatment induces changes in the level expression of macrophage MHC II. Objective: To quantify the level of expression of MHC II in macrophages using real-time PCR; and immunofluorescence detection of membrane bound MHC II using fluorophore-conjugated antibodies and confocal microscopy.
• Determine whether treatment with Zwitterionic Polysaccharide-based TACA Delivery approach induces changes in macrophage morphology indicative of immune activation. Objective: To monitor changes in macrophage morphology in vitro (e.g. morphological changes known to be associated with transition to an M1 phenotype) using phase-contrast microscopy

           

 

3. Significance

            The present study aims for providing appropriate information regarding the tumor associated carbohydrate antigen as a potential cancer vaccine. The existing literature and the literature review represented in this chapter has described the type of imperative methods that has been developed for combating the serious threats of cancer. The information concerning the TACA vaccines and immunotherapy against cancer have been developed for the prevention and treatment of cancer.  The development of these therapeutic cancer vaccines has been found to be quite efficient in eliminating the tumors and blocking the activity of the malignant cells from converting into tumor cells. Hence, these vaccines have been effective in boosting the immune response against the tumor cells and prevents the body cells from any type of tumor. The development of tumor-associated carbohydrate antigens (TACAs) has evolved as an important biomarker for the cancer prognosis which is officially declared by the National Institutes of Health (Feng et al., 2016). The association of TACA have been conjugated with T-cells that stimulates protein carriers evolving keyhole limpet haemocyanin (KLH), tetanus toxoid (TT), bovine serum albumin (BSA), and diphtheria toxin (CRM197) that further shows more efficiency towards tumor cells (Guo & Wang, 2009). The previous form of TACA was found to be efficient but with the advancement of these protein carriers, the vaccine has been found to be self-immunogenic while suppressing the antigen-specific immunogenicity (Nativi&Renaudet, 2014). Also, the coupling of TACAs with polysaccharides, often known as zwitterionic polysaccharide, PS A1, toll-like receptor 2 ligand, T-cell peptide epitopes have been found to show efficacy towards the development of self-adjuvating and multi-component cancer vaccines (Shi et al., 2016; Patronov&Doytchinova, 2013; Lakshminarayanan et al., 2012). The development of these vaccine antigens has been effective in the treatment of phase II prostate cancer that mostly involved vaccines such as a hexavalent vaccine construct with incorporation of GM2, globo H, Ley, clustered Thomsen nouveau (Tn), clustered Thomsen-Friedenreich (TF), and glycosylated mucin 1 (MUC1) (Slovin et al., 2007). Hence, it is said that the complex nature of carbohydrates has been proved to be effective for the diagnosis of cancer along with the development of therapeutic vaccines that kill tumor cells effectively. The available carbohydrate dependent antigens have shown quite effective results to combat the threats of cancer. The association of TACA with other protein carriers have been effective in the production of new vaccines that overcome the immunosuppressive nature of carrier proteins. Hence, this proves to be effective in generating immune response against the tumor cells.

 

4. Research Methodology

            The present study aims for analyzing the role of tumor associated carbohydrate antigen as a potential cancer vaccine. To fulfill this aim, the present study has adopted the concept of formulating a Zwitterionic Polysaccharide capsular protein needed for preparing off shelf vaccines to do in vitro in vivo tests. Therefore, this study has adopted the Zwitterionic Polysaccharide-based TACA Delivery approach for fulfilling the major aim of the study.

 

4.1 Cell Culture

            The present research will use commercial cell lines of animal models along with human monocytes and human tumor models. These animal cell lines will be maintained in DMEM media, human monocytes in RPMI-1640 and human tumor cell lines in FBS.

4.2 Zwitterionic Polysaccharide capsular protein Extraction

Zwitterionic Polysaccharide capsular protein will be extracted as per the methodology of Ziegler et al., 2014.

4.3 Experimental Conditions

            Macrophages will be harvested. The in vivo and in vitro experiments will be carried out.

4.4 Nitrate Assay

A nitrite standard curve will be generated using sodium nitrite dissolved in fresh culture media.

4.5 Enzyme linked immunosorbent assay (ELISA)

            Elisa will be performed on cell culture media following the treatment of cells to detect secreted cytokines.

4.6 RNA Extraction

RNA extraction will be performed using the RNeasy Mini kit.

4.7 cDNA Synthesis

All steps for cDNA synthesis will be undertaken in accordance with the manufacturer’s supplied protocol.

4.8 Primer Design     

Sequence specificity of primers will be all verified using PrimerBLAST.

4.9 Real-Time PCR

Relative levels of gene expression will be measured using thermocycler.

4.10 Immunofluorescence

4.10.1 MHCII expression

The protocol for the same will be followed and used. Confocal microscopy will be used for analyzing the images.

4.11 Phase Contrast Microscopy

Phase contrast microscopy will be performed using an inverted microscope to observe morphological changes within macrophages upon exposure to immunostimulatory molecules.

4.13 Statistical Analyses

All samples will run in triplicate and presented as the mean ± SE of the mean (SEM) unless otherwise specified. One-way ANOVA and Tukey’s post-hoc statistical tests will be used to analyse all observed results for statistical significance.  All data will be subjected to Kolmogorov-Smirnov (KS), Shapiro-Wilk, and Anderson-Darling tests to ensure a normal distribution. Linear and nonlinear ANOVA trend analyses will be used to evaluate dose relationships. All statistical analyses will be performed using the SPSS.

 

 

 

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