Production and characterization of monoclonal antibody against a triple negative breast cancer cell line

https://doi.org/10.1016/j.bbrc.2018.09.087Get rights and content

Highlights

  • Discovery of potential biomarkers that can predict tumor behavior is essential in breast cancer.

  • A panel of monoclonal antibodies was produced by hybridoma technology.

  • MAbs that reacted with surface antigens of immunizing cell line but not normal cells were selected.

  • Target was identified as NCAM1 by immunoprecipitation and mass spectrometry.

  • The anti-NCAM1 antibody has potential application in cancer diagnosis.

Abstract

Breast cancer is the most prevalent malignancy among women around the world such that more than 1,400,000 new cases are being diagnosed each year. Despite immense studies over many years on diagnosis and treatment of breast cancer, about 30% of treated patients will relapse and require subsequent therapy. By development of hybridoma technology, murine monoclonal antibodies (MAbs) against several human tumor-associated antigens have been produced and characterized in many laboratories. The purpose of these studies is to generate effective monoclonal antibodies that could be useful in tumor diagnosis and therapy. In this study, splenic lymphocytes of immunized BALB/c mouse with a new established breast cancer cell line (Pari-ICR cell line, established in Shiraz Institute for Cancer Research) were fused with the mouse myeloma cell line SP2/0 in the presence of polyethylene glycol. We generated a panel of monoclonal antibodies against the newly established cell line. The hybrid cultures were screened by flow cytometry. Hybridomas that produced antibody to surface antigens of immunizing cell line but not to Human Gingival Fibroblasts, adipose stem cells, and leucocytes isolated from peripheral blood were selected and cloned by limiting dilution method. The 1E3 clone (IgG2a type) that displayed clonal stability was further analyzed for specificity by flow cytometry. MAb 1E3 showed weak to strong reactivity to other cell lines compared with Pari-ICR cell line. Antigen identification was performed by a workflow consisting of immunoaffinity purification, SDS-PAGE, Western blotting, and mass spectrometry analysis. The target of 1E3 mAb was identified as NCAM1. In conclusion, using the antibody-based strategy we identified NCAM1 as a potential therapeutic target and biomarker for breast cancer.

Introduction

Due to rising cancer incidence over the time, its global importance is undeniable. Breast cancer is the most prevalent malignancy among women around the world such that more than 1,400,000 new cases are being diagnosed annually. This disease is caused by a combination of genetic and environmental factors. Despite immense studies over many years on diagnosis and treatment for breast cancer, about 30% of treated patients will relapse and require subsequent therapy [1,2].

Since the identification of new disease-specific biomarkers can accelerate the drug discovery, many studies have been conducted in order to identify biomarkers as new targets for effective diagnosis and cancer therapy. Nowadays, several tumor markers are being utilized for screening, staging, prognosis, treatment, and surveillance of breast cancer including Carcinoembryonic antigen (CEA), CA 15–3, CA 27.29, Estrogen receptor (ER), Progesterone receptor (PR), Human epidermal growth factor receptor 2 (HER2), Plasminogen activator inhibitor1 (PAI-1), and Urokinase plasminogen activator (uPA) [3,4].

During the past decades, genomics and proteomics techniques have introduced an enormous number of candidate tumor-associated antigens. Since the alternative splicing of mRNA, along with post-translational modifications, causes each gene to give rise to different possible protein products, many of targets cannot be simply recognized by genomic approaches. In addition, there are many context-dependent cancer-specific epitopes that are only presented on the whole living cell in the context of other membrane-associated macromolecules. Identification of these epitopes by genomics-based and proteomics-based approaches involves high difficulty. Therefore, other techniques must be used to identify these cancer-related antigens [5,6].

The monoclonal antibody-based technologies provide an approach to complete genomics and proteomics attempts in which intact cells are used as a source of antigens for detection of cancer-specific cell surface targets. With this technology, a panel of monoclonal antibodies (mAbs) with a high level of epitope diversity is produced [7].

In the present study, hybridoma cells were produced by immunizing mice with a Pari-ICR cell line obtained from one patient with breast cancer. MAb that reacted with a surface antigen of immunizing cell line, but not normal cells, was selected and the target was identified by immunoprecipitation and mass spectrometry.

Section snippets

Cell culture

Cancer cell lines (MDA-MB-231, MCF-7, SKOV3, MIA-PaCa-2, MDA-MB-468, PaTu 8902, and mehr80) and normal human fibroblast were purchased from National cell Bank of Iran based in Iran Pasture Institute; ASCs were extracted from breast adipose tissues of healthy donor with cosmetic mammoplasty surgery after obtaining written informed consent and characterized as described before [8]. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. Normal human fibroblast and ASC were cultured

Generation and selection of mouse monoclonal antibodies against Pari-ICR cell line

In this study, a new established triple negative breast cancer cell line (Pari-ICR) isolated from the primary breast tumor of a 36 years old patient with grade II invasive ductal carcinoma was used for immunization. The mouse showing the highest titer was sacrificed and splenocytes were fused with SP2/0 cells for hybridoma production. After fusion, screening of growing hybridomas was performed by flow cytometry. According to the obtained results, 5 out of 78 antibody-producing hybridomas showed

Discussion

Breast cancer, one of the most controversial malignancy, is the second most common type of cancer after lung cancer (10.4% of all cancer incidence, both sexes counted), for which no significant progress in prognosis and patient survival has been reported [9,10]. Therefore, the discovery of other potential biomarkers that can predict tumor behavior is of great importance in breast cancer. The antibody-based approach provides an opportunity to find out new targets and target epitopes with

Conclusion

Using the antibody-based strategy, we identified NCAM1 as a potential therapeutic target and biomarker for breast cancer. Monoclonal Anti-NCAM1 antibody that was produced in our laboratory is suitable for Western blotting, flow cytometry, and immunohistochemical analyses. The produced monoclonal antibody may be against a novel epitope of NCAM1. In order to evaluate the specificity of the antibody, we need screening more normal and cancer tissues. Additionally, since there are specific breast

Acknowledgments and funding

This work was financially supported by a grant from Shiraz university of Medical Sciences (grant number: 94-7652) and in part by Shiraz Institute for Cancer research (grant number: ICR-100-508).

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