In silico designing and expression of novel recombinant construct containing the variable part of CD44 extracellular domain for prediagnostic breast cancer

Abstract Background CD44, as a tumor‐associated marker, can be used to detect stem cells in breast cancer. While CD44 is expressed in normal epithelial cells, carcinoma cells overexpress CD44. Aims In the current study, we designed a recombinant protein that included the variable component of the CD44 (CD44v) extracellular domain to apply in clinical diagnosis of breast cancer. Methods A total of 100 CD44v amino‐acid residues were determined, and the structure was examined using bioinformatics tools. The construct was inserted into the PET28a vector and transformed in E. coli BL21(DE3). A nearly 12 kDa fusion protein was obtained by Ni‐NTA affinity metal chromatography. Recombinant CD44v was examined by Western blotting, ELISA, and immunohistochemistry (IHC) assays. Results The findings revealed that the structure of rCD44v was stable, and its antigenic domain was exposed. The recombinant CD44v was confirmed by western blotting, and the presence of antibodies against recombinant CD44v protein in the patient's serum was detected by the ELISA. Our data demonstrated a link between CD44v serum levels and the prevalence of breast cancer. Conclusion Assessments of antiCD44v antibodies with rCD44v could be a useful tool for identifying breast cancer in its early stages, which can lead to better outcomes.

source of cancer establishment, leading to cancer reappearance and metastasis years after cure. Self-renewing cells are a tiny minority of tumor cells that may split into multiple cell types, according to recent research. 4,5 Cancer stem cells can start a tumor and cause the disease to progress in a limited number of breast cancer populations. In addition, resistance to chemotherapy and radiation contributes to treatment failure and disease relapse; hence, identifying and monitoring BCSCs plays a critical role in prognosis and treatment resistance, potentially opening up new therapeutic options. 1,6 Many efforts are currently being undertaken to improve advanced diagnostic and therapeutic procedures as well as develop new super tools, such as discovering markers for BCSCs. Important surface indicators in cancer stem cells have recently been found in several investigations. 1 BCSCs in breast cancer accounts for roughly 2% of all tumor tissue. As a result, getting these cells out of tumor tissue is tough. Many researchers have been hunting for surface breast cancer antigens by identifying the subgroup of BC cells that express CD44 + /CD24 À as a distinctive hallmark of BCSCs. 1,7 Furthermore, the CD44 CSC surface marker interacts positively with initiator cells. 8 As a result, the introduction of a complete diagnostic probe for CD44 can lead to early diagnosis and improved treatments. 5 Antibodies or autoantibodies that can react with tumor cells, tissue, and isolated proteins are generated concurrently with the development of tumor-dependent antigens, while they are scarcely visible at the early stage. Antibodies, on the other hand, are physiologically increased and quantifiable in the early stages of breast cancer, giving them a viable tool for identifying cancerous tissue from healthy tissue. 9,10 CD44, which is recognized either as a surface marker of cancerinitiating cells (CICs) or cancer stem cell antigen, was utilized as a key marker for identifying BCSCs, linking with tumor aggressiveness, metastasis, and recurrence. 5,11,12 CD44 is a non-kinase transmembrane glycoprotein having a cytoplasmic carboxyl-terminal tail and extracellular and transmembrane domains.
The CD44 gene has 19 and 20 exons in humans and mice, respectively; the exon number six and the variant number one have no similarity in humans, while the first and last five exons are fixed in all isoforms and encode the shortest isoform of CD44, known as standard CD44 (CD44s). Furthermore, central exons can be alternatively cleaved and constructed with the 10 exons found in the conventional CD44 isoform, known as "variables," associated with CD44 variant (CD44v) isoforms, which include the middle nine exons. To make a single variant exon, CD44v isoforms can be coupled with other variant exons that code for extracellular domain peptides. 13,14 Different CD44v isoforms affect the structure of cell surface as well as the receptors for cytokines and growth factors. CD44's biological activity is influenced by these distinct binding motifs. Hence, cancer cells frequently express a large number of CD44 variations, particularly in the late stages of progression. 11,13,14 Cell-cell and cellsubstrate interactions, such as proliferation, differentiation, cell migration, cell adhesion, signaling, and survival, are all aided by CD44.
Meanwhile, proteolytic enzymes may release this molecule as a cell surface glycoprotein into circulation. As a result, the amount of soluble CD44 proteins in cancer patients' serum correlates with tumor growth and metastasis in various carcinomas. [14][15][16] CD44 has a high level of expression on bulk tumor cells and CICs, while it has a low level of expression in healthy tissue and differentiated breast tissue, according to the findings.
Up-regulation of CD44, according to immunohistological research, influences the pace of cell invasion, tumor growth, and metastasis in a variety of malignancies, including breast cancer. Furthermore, patients with CD44 overexpression have had unfavorable outcomes.
Breast cancer treatment frequently has favorable results when compared to other types of early-stage cancer treatment, making this initial identification critical, and CD44 is the diagnostic target in the early stages of the disease. 1,5 We assumed that BCSC biomarkers are visible in patients' serums because the most extensively utilized biomarkers, HER2 and MUC1, are employed in breast cancer.
Because of the heterogeneity created by the complexity of expression CD44v isoforms, it is critical to screen breast cancers and employ these isoforms as diagnostic biomarkers. CD44v was employed as a BCSC marker in this study to look at the link between serum levels and pathological variables. Our objective was to create a recombinant antigen containing a short common section of variable area exons the extracellular domain (rCD44v) that may be utilized as a coating antigen in immunoassay methods to detect anti-CD44v antibodies. Also, in this study, we showed that polyclonal antibodies obtained from mice are able to detect CD44v antigen in the serum of breast cancer patients by ELISA method, and we further determined that above antibodies have demonstrated this property in patient's tissue by immunohistochemistry.

| Cases and controls
A total of 67 tissue and serum samples were taken, 30 healthy women with an average age of 43 and an age range of 26 to 60 years, four patients with benign breast tumors with an average age of 37, and an age range of 26 to 49 years, and 33 breast cancer patients with an average age of 49 and an age range of 28 to 71 years, before and after treatment including surgery, post-operative radiotherapy, and, or chemotherapy from Imam Khomeini Hospital (Tehran, Iran). We  (Table 1).

| Expression and purification of CD44v
To create the pET28-CD44v construct, the 107-amino-acid designed sequence was synthesized and cloned into the pET28a (General biosystem service). GeNetBio kit was used to extract plasmids (Catalogue # K-1000). A PCR reaction with T7 universal primers was carried out to validate the pET28a-CD44 construct. Furthermore, the sequence of construct was presented and confirmed by General biosystem service. The construct was used to convert E. coli BL21 (DE3) competent cells using a thermal shock technique. 20 Four milliliter of pET28a-CD44 harboring E. coli BL21 (DE3) was inoculated into 200 ml LB broth encompassing 25 μg/ml kanamycin (Sigma-Aldrich, Catalogue # K1637) and incubated at 170 rpm and 37 C for 2 h to reach OD600 to 0.7-0.9, then 1 mM IPTG (Sinaclon, Catalogue # CL5812) was added. The six His-tagged fusion protein of CD44 (rCD44v) was subjected to Ni-NTA column chromatography (Qiagen, Catalogue # 30210) under native conditions. 21 Different concentrations of imidazole(Merck, Catalogue # 104716) followed by a pH gradient procedure were used to elute the rCD44v antigen. Non-induced recombinant cells were considered as the negative control; 15% SDS-PAGE was used to check the purity, and protein content was estimated by the Bradford method. 22

| Recombinant protein confirmation by western blotting
The purified antigen was electrophoresed on gel 15% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane using transfer buffer. After running, the membrane was blocked by 5% BSA (Sigma, Catalogue # 7030) at 37 C for 1 h. Thereupon, the PVDF membrane was washed three times with 0.05% tween 20 in PBS buffer (PBST). The recombinant CD44v was incubated with a mouse antibody against His-tag conjugated with HRP (1/2000, Roche, Catalogue # 11965085001) in PBST buffer and was shaken at 37 C for 1 h and then washed with PBST buffer several times. In the next step, the detection was performed using an HRP staining solution (DAB) (Diagnostic Biosystems, Catalogue # K047). The chromogenic process was finally stopped by washing twice with pure water. 23

| Preparation of anti-CD44v serum
Twenty micrograms of recombinant CD44v protein was injected subcutaneously into the back of necks of 5-week-old female BALB/c mice with complete Freund's adjuvant (Sigma, Catalogue # F5881).
On days 14 and 28, an incomplete Freund's adjuvant (Sigma, Catalogue # F5506) was utilized as a booster dosage in future injections. Furthermore, as a negative control, full and incomplete Freund's adjuvant was administered in the same way as previously. The indirect ELISA technique was used to detect antibodies against the rCD44v antigen, and then the mouse serum was collected and kept at À70 C for future usage. 24

| Immunohistochemical analysis
The prepared mouse polyclonal antibody (pAb) against rCD44v and monoclonal antibody (mAb) against hCD44 (DAKO, Catalogue # M and incubated for 1 h at 37 C, followed by the addition of OPD as a peroxidase substrate, the color was developed, and OD492 was recorded. A standard curve was created to measure the relative values of rCD44v in serum samples, and the optical density of CD44v antigen in patients' serum was measured using the standard curve.

| Statistical analysis
The data are obtained from three independent experiments and analyzed using SPSS version 17.0 (SPSS Inc.), as mean ± standard deviation. For multigroup comparisons, the statistical significance was determined using the Two-way ANOVA test (p-value >.05). F I G U R E 3 rCD44v-specific serum IgG following subcutaneous immunization. Mice were injected with recombinant CD44v using complete and incomplete Freund's adjuvants. Immunizations were performed three times within 6 weeks. The sera were collected after immunization and assessed for rCD44v-specific IgG by the ELISA method. Non-immunized mice sera were used as control (p < .05).

| Structural design and prediction
(441-540) was chosen for assembly in this investigation. As a fusion tag, six His amino acids were utilized at the C-terminus ( Figure 1A).

| Other structural features
Algpred tool and SDAP allergen library were applied to predict the allergenicity of sequences. According to their results, there is no potential for allergenicity of rCD44v antigen. Table 2 summarizes the primary epitope properties, including hydrophilicity, accessibility, stability, antigenicity, polarity, and exposed recombinant protein surfaces. Table 3 also displays the anticipated B and T cell epitopes with the greatest interaction score for both MHC Classes I and II.

| Expression, purification of rCD44v protein
The PCR reaction was performed with T7 promoter primer and T7 terminator  Figure 2C). In the negative control, however, there was no reactivity ( Figure 2C).

| Detection of specific mouse Abs against CD44v with ELISA and IHC
Immunization of mice with pure rCD44v protein resulted in the formation of specific IgG antibodies. Compared with mice control antisera, an anti-rCD44v IgG antibody titer can be detected even at 1/1600 dilutions (Figure 3). Our findings revealed that rCD44v can be detected by either anti-CD44 or anti-His-tag antibodies. Furthermore, IHC examination exhibited that the mice anti-CD44 polyclonal Ab, like typical anti-CD44 monoclonal Ab, can detect CD44 protein expressed in breast tumors (Figure 4).

| Detection of CD44v protein in patients' sera
ELISA analysis revealed that rCD44v protein was capable of detecting anti-CD44v antibodies in patients' serum. This means that natural anti-CD44v antibodies can recognize epitopes on rCD44v ( Figure 5A).
In addition, anti-rCD44v antibody identified CD44v proteins in patients' serum on the plate ( Figure 5B and Table 1).  Although there has been a negative association between circulating anti-CD44 antibodies and antigen in patients with specific malignant tumors, CD44 antigen exhibited a substantial negative link with the matching antibody in breast cancer patients in our current investigation.

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Meanwhile, there is no correlation between HER2 and CA15-3 levels and CD44, based on patient characteristics (Table 1) and our findings. [29][30][31] Our findings suggest that low and high CD44v antigen levels in individual patients may be linked to low and high anti-CD44v antibody levels, respectively. Synchronized elevation of CD44v antigens and anti-CD44v antibodies in serum can be expected when breast cancer has progressed to a late stage. As a result, anti-CD44v antibodies may bind to CD44v antigen in breast cancer patients, implying that recurrence or distant metastasis may play a role in CD44v antigen overexpression. 13,27,29 To summarize, we used two ELISA techniques to look for benign and malignant breast cancers in blood samples. Recognizing the CD44v antigen, according to our findings, is required in breast cancer, particularly in malignant tumors. In addition, assessing the quantity of anti-CD44v antibodies in the blood can be a useful tool for identifying breast cancer in its early stages, which leads to better outcomes. To expand the study, further extensive investigation with more instances is required.

ACKNOWLEDGMENT
The authors appreciate the support provided by the Research and

CONFLICT OF INTEREST
The authors have stated explicitly that there are no conflicts of interest in connection with this article.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
The present study was approved by the Ethics Committee of

CONSENT TO PARTICIPATE
Informed consent was obtained from all individual participants included in the study.