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

Abstract

Monoclonal antibody (mAb) technology is an excellent tool for the discovery of overexpressed cell surface tumour antigens and the development of targeting agents. Here, we report the development of two novel mAbs against CFPAC-1 human pancreatic cancer cells. Using ELISA, flow cytometry, immunoprecipitation, mass spectrometry, Western blot and immunohistochemistry, we found that the target antigens recognised by the two novel mAbs KU44.22B and KU44.13A, are integrin α3 and CD26 respectively, with high levels of expression in human pancreatic and other cancer cell lines and human pancreatic cancer tissue microarrays. Treatment with naked anti-CD26 mAb KU44.13A did not have any effect on the growth and migration of cancer cells nor did it induce receptor downregulation. In contrast, treatment with anti-integrin α3 mAb KU44.22B inhibited growth in vitro of Capan-2 cells, increased migration of BxPC-3 and CFPAC-1 cells and induced antibody internalisation. Both novel mAbs are capable of detecting their target antigens by immunohistochemistry but not by Western blot. These antibodies are excellent tools for studying the role of integrin α3 and CD26 in the complex biology of pancreatic cancer, their prognostic and predictive values and the therapeutic potential of their humanised and/or conjugated versions in patients whose tumours overexpress integrin α3 or CD26. Pancreatic cancer remains one of the deadliest cancer types. In 2018, there were an estimated 458,918 new cases of pancreatic cancer, and 432,242 deaths as a result of pancreatic cancer in 185 countries worldwide 1,2. Pancreatic cancer is predicted to become the second leading cause of cancer death after lung cancer, within the next decade in Western countries 3. At present, the only curative treatment for patients with pancreatic cancer is surgery. However, only a minority of patients are eligible for resection and disease recurrence is a frequent event in many such patients. Historically, gemcitabine-based therapy has been the mainstay for treatment of pancreatic cancer 4. More recently the combination of gemcitabine plus capecitabine has been regarded as the new standard of care in the adjuvant setting 5. Patients with metastatic disease are treated with either FOLFIRINOX or gemcitabine plus nab-paclitaxel as first-line in patients with good performance status 6,7. In order to reduce the dismal pancreatic cancer mortality rates, it is essential to discover novel biomarkers for use in the early detection of pancreatic cancer, to discover novel therapeutic targets and to develop novel and more effective therapeutic agents 8,9. Monoclonal antibodies (mAbs) are excellent tools for the discovery of novel overexpressed cell surface antigens and their specific targeting for diagnostic and therapeutic purposes 10,11. To date, 36 mAbs have been approved for cancer treatment in the U.S. and/or European Union, although none for pancreatic cancer yet 12,13. As tumour heterogeneity has been reported both between (i.e. inter-tumour heterogeneity)

<p>CD26 as a Promising Biomarker for Predicting Prognosis in Patients with Pancreatic Tumors</p>

“…12 Furthermore, Arias-Pinilla et al have shown that 61% of the 34 tissue samples expressed CD26. 13 These two studies are consistent with our results. Here, we assessed CD26 expression using a larger sample size and analyzed in detail its correlation with various clinicopathological features.…”

Therapeutic Application of Monoclonal Antibodies in Pancreatic Cancer: Advances, Challenges and Future Opportunities

“…The results from cell proliferation assays in human pancreatic cancer cell lines, cell line-based xenografts and patient-derived tumour xenografts have demonstrated antitu-mour activity either as single agents or in combination with cytotoxic drugs. The results of in vitro studies have supported the therapeutic potential of mAbs targeting integrin α3 [26], MUC4 [27], MUC1 [28], and MUC13 [29] when used alone or in combination with cytotoxic drugs. Furthermore, mAbs targeting EGFR, TROP2 and α6β4 have been used for radioimmunotherapy and have shown effective localisation of primary tumours and metastatic sites in mouse models [30][31][32].…”

“…Indeed, using monoclonal antibody technology and human pancreatic cancer cell lines established from primary tumours and metastatic sites, both as the source of tumour immunogen and in the antibody screening, we have reported recently the development of three novel antibodies. We found that the antigens recognised by these three novel mAbs were CD109, integrin α3 and CD26, with high levels of expression in several human pancreatic cancer cells and Pancreatic Cancer Tissue microarray [26,66]. Indeed, antibody-based screening will help not only in the discovery of additional therapeutic cell surface antigens with high levels of expression at different stages of pancreatic cancer (i.e., therapeutic targets) but also in the development of antibody-based agents for therapy.…”