An In silico Chimeric Vaccine Targeting Breast Cancer Containing Inherent Adjuvant

Breast cancer is one of the most common causes of death in women all around the world. It is estimated that more than 1.38 million new women suffered from breast cancer (1). Anti-estrogen therapy is the choice strategy for estrogen receptor positive breast cancer patients. However, a large number of breast cancers do not respond to this type of treatment because of lack of estrogen receptor. Furthermore, another group of breast cancer becomes insensitive to hormone therapy after first therapy and immediately promotes to grow (2). Although chemotherapy is an alternative strategy for treating insensitive and metastatic breast cancer, but many of the treated cancers often develop a recurrence. Therefore, innovating novel agents is an urgent requirement for attenuating the mortality rate (3). The receptor tyrosine kinase like orphan receptor 1 (ROR1) is a transmembrane protein and belongs to the receptor tyrosine kinase family (4). This protein has five domains, three extracellular including an immunoglobulin like motif, frizzled and Kringle domains, transmemebrane part and an intracellular tyrosine kinase domain (5). Over expression of ROR1 is found in the embryonic stage (6) and several cancers including B-CLL (7), B-ALL (8), gastric carcinoma, non-small cell carcinoma cell lines (9) and breast cancer (10). Zhang et al. revealed the overexpression of ROR1 in the breast cancer related with lacked of HER2/neu and hormone receptors. Moreover, upregulation of ROR-1 in primary breast cancer is associated with poor differentiation and shorter survival rate (10). There are limited or lack of ROR-1 in normal cells while over expression of this protein has been reported among human cancer cells, therefore ROR1 may be applied as a potent target for immunotherapy.

Staphylococcal enterotoxin B (SEB), a 28 KDa superantigen, is a powerful T cell activator. This protein binds to MHC class II on antigen presenting cells (APCs) and then forms complex with the variable region of β chain of T cell receptor. The binding site of SEB on the APCs has differed from that of specific antigens (11). SEB exerts a potent mitogenic effect on both CD4+ and CD8+, increasing cytokines including interferon-γ (INF-γ), interleukin 2 (IL-2), and tumor necrosis factor-α (TNF-α), finally promotes a powerful antitumor immunity (12).

One of the main goals of immunotherapy against tumor is to create a specific tumoral antigen response that participates to the tumor eradication. Designing the combined construct, enabling to activate both cellular and humoral anti-tumor immunity is an effective therapeutic method which restricts or eradicates tumor progression. To date, it is possible to design a suitable combined construct, based on B cell and the T cell epitope map using bioinformatics methods.

Here, we purposed to design the immunotherapeutic target constructed having two parts. The first one was extracellular domains of ROR1 as specific tumoral antigens for inducing B cell lymphocyte and second one is SEB, an adjuvant to create anti-tumoral response via T cell lymphocyte.

Development of efficacious therapeutic strategy for some of the resistant malignancies is the main emergency of health organization all around the world. Nowadays, design of appropriate and safe vaccines which stimulate the immune response actively or passively, are the hot topic in the field of reverse vaccinology. This area is closely related to computational vaccinology that recruits discrepant informatics tools to predict efficient T- and B- cell functional epitopes to improve the properties of an antigen based vaccine (24, 25).

The principal purpose of the current study was to design a unique construct, including two antigenic parts, which adjoined together through hydrophobic linker (26, 27). Theoretically, our structural model could augment immunogenicity of ROR-1 protein and owing to the presence of staphylococcal enterotoxin B as a potent superantigen; its probability evokes a wide cellular or humoral anti-tumor immune response. As respects to the momentous role of linker in representing the pattern of various epitopes throughout the chimeric protein besides maintenance of its functional properties, the linker selection is a key point in designing of the fusion protein (26). In this study a flexible linker, GSGGSGGSGGSG With 12aa, was used to separate domains of two proteins. Aria et al. repotted the multimerizing property of short helical linker compared with longer ones. Furthermore, a flexible linker based on shorter conformation plays an efficient role in comparison to those with the helical linker (26). To predict the secondary structure of a chimeric protein, the GOR method was applied. This software allows estimating the possible secondary structure of each amino acid together with its impact on the condition and structure of adjacent amino acids. The most abundant structure within our fusion protein was a random coil that could be due to the presence of a high amount of hydrophobic amino acids such as glycin. In accordance with finding from the physico-chemical parameter analysis, our fusion protein had an acidic nature with the high extinction coefficient at 280 nm, which is owing to high content of Cys, Trp and Tyr. In contrast to partial instability of our fusion protein, its estimated high alphabetic index infers to protein stability in a broad range of temperature.

One of the most important problems in the designing of recombinant protein is the biologic functional characteristics. Although prediction of secondary structure by ab-initio methods or folding recognition is able to detect some of the limitations (28), prediction of three- dimensional structure through comparative and ab-initio methods attenuates several errors (29, 30). The three-dimensional model of the fusion protein ROR-1-SEB protein was accounted using the I-TASSER server (14) according to their confidence score (C-score), Z-score, RMSD and TM-score. This server suggests five models for our chimeric sequence that model 2 had more c-score between them, therefore it selected for further evaluation. Expected TM-score obtained 0.53 ± 0.15, which accredited the validity of the model. A TM-score more than 0.5 portend accuracy of topology. Data from Procheck Ramachandran plot demonstrated the stability of the fusion protein. Thereabout, 8.7% of the residues located in outline region, which, could presumably be owing to fusion. Since the purpose of designing a vaccine is the generation and selection of a candidate with the potential stimulation of strong responses (31), we analyzed the epitope maps for B cell and T cells. At first, we predicted linear B-cell epitope overall the chimeric sequence using BCpreds. Moreover, for recognizing the epitopes involved in antibody-antigen interaction, the estimation of conformational epitopes is an essential in the computational vaccine design which executed using both structure and sequence information based method, including DiscoTope and CBTOPE, respectively. Our result showed the copious b-cell epitopes, though some of the solely predicted by one method. In order to predict the map of T-cell epitope and binding affinity to both classes of MHC molecules, Propred and Propred-1 were applied. Numorous T-cell epitope with a high antigenicity score were suggested by two methods, but we only selected those epitope that is simultaneously proposing as B-cell and T-cell epitope (Table 6). Our result suggested that our structural model represented the epitope that are capable to be a stimulant for both T-cell and B- cell mediated immune responses. At last, this structure showed no significant resemblance with the allergen in the SDAP library.