Development of novel monoclonal antibodies against CD109 overexpressed in human pancreatic cancer

Abstract

Pancreatic cancer is one of the most aggressive and lethal types of cancer, and more effective therapeutic agents are urgently needed. Overexpressed cell surface antigens are ideal targets for therapy with monoclonal antibody (mAb)-based drugs, but none have been approved for the treatment of pancreatic cancer. Here, we report development of two novel mouse mAbs, KU42.33C and KU43.13A, against the human pancreatic cancer cell line BxPC-3. Using ELISA, flow cytometry, competitive assay and immunoprecipitation followed by mass spectrometry, we discovered that these two mAbs target two distinct epitopes on the external domain of CD109 that are overexpressed by varying amounts in human pancreatic cancer cell lines. Treatment with these two naked antibodies alone did not affect tumour cell growth or migration in vitro. Of the two mAbs, only KU42.33C was useful in determining the expression of CD109 in tumour cells by Western blot and immunohistochemistry. Interestingly, immunohistochemistry of human pancreatic carcinoma tissue arrays with mAb KU42.33C showed that 94% of the 65 human pancreatic adenocarcinoma cases were CD109 positive, with no expression in normal pancreatic tissues. Our results suggest that these two novel mAbs are excellent tools for determining the expression level of CD109 in the tumour specimens and sera of patients with a wide range of cancers, in particular pancreatic cancer, and for investigating its diagnostic, prognostic and predictive value. Further research is warranted and should aim to unravel the therapeutic potential of the humanised forms or conjugated versions of such antibodies in patients whose tumours overexpress CD109 antigen.

Development and application of two novel monoclonal antibodies against overexpressed CD26 and integrin α3 in human pancreatic cancer

“…The exquisite specificity of the antibodies for the target antigen also makes them ideal tools for investigating the biological, diagnostic, prognostic and predictive value of their target antigens and for use in targeted therapy of human cancers 10,11,23,24 . We have previously reported the generation of two novel anti-CD109 mAbs by means of hybridoma technology using BxPC-3 human pancreatic cancer cells, a cell line derived from a primary tumour, as the source of immunogen 9 . However, human cancers are heterogeneous in nature, a characteristic feature which contributes to the response of short duration or development of resistance to current therapies 21 .…”

“…formed at St George's University of London, following ethical approval and under Home Office animal license as described previously 9 . All experiments were performed in accordance with relevant guidelines and regulations.…”

“…Antibodies secreted from novel hybridomas were screened by ELISA as described previously 9 . Newly formed positive hybridomas were selected, cloned twice by limiting dilution technique, adapted to growth medium containing 3% FBS, grown in roller bottles and the hybridoma supernatants were harvested and purified for further studies as described below.…”

“…We reported recently the development of two novel antibodies against an antigen with high level of expression in pancreatic cancer (i.e. CD109) using the human pancreatic cancer cell line BxPC-3 (derived from a primary tumour) as the source of tumour immunogen 9,16 . BxPC-3 is a moderate to poorly differentiated cell line derived from a 61-year-old Caucasian female with a primary body of pancreas adenocarcinoma in whom no metastatic disease was found and who died 6 months later despite chemotherapy and radiation 16 .…”

CD109: a multifunctional GPI‐anchored protein with key roles in tumor progression and physiological homeostasis

“…High levels of CD109 expression have been detected in various tumor‐cell lines and tumor tissues, including those associated with squamous cell carcinomas (SCCs) of the lung, esophagus, uterus, and oral cavity, adenocarcinomas of the lung and pancreas, breast cancer, glioblastoma, hepatocellular carcinoma, urothelial carcinoma of the urinary bladder, and several types of sarcomas (Table ) . Immunohistochemical (IHC) analyses revealed elevated CD109 expression on tumor cells (Fig.…”