Arias Pinilla, Gustavo (2017) Development and characterisation of novel monoclonal antibodies against human pancreatic cancer cells for use in cancer diagnosis and therapy. (PhD thesis), Kingston University, .
Abstract
Pancreatic cancer is the 12th most common diagnosed cancer and the 7th leading cause of cancer death worldwide with a median survival of approximately 6 months and a five-year survival rate of only 3-5%. Despite some advances in diagnosis and treatment, the survival rates have not improved considerably over the past four decades. Targeted therapy has emerged as an alternative for the treatment of various cancer types and monoclonal antibodies (mAbs) therapy has become an attractive therapeutic approach due its specificity and reduced toxicity in comparison with conventional forms of treatment (chemotherapy and radiotherapy). Although some mAbs have been approved for treatment of haematological malignancies and solid tumours, none have yet been approved for pancreatic cancer. The aim of this study was to identify over-expressed cell surface antigens of biological significance in pancreatic cancer using hybridoma technology and to investigate their potential as therapeutic targets for antibody-based immunotherapy. A panel of 11 novel monoclonal antibodies against over-expressed antigens on human pancreatic cancer cells was developed. These mAbs were found to be directed against ten different antigens/epitopes based on the patterns of binding to a panel of cancer and non-cancer cell lines. Of these, seven mAbs (KU42.33C, KU42.77, KU43.13A, KU44.2, KU44.4C, KU44.22B and KU44.71A) are lgG1 Kappa isotype while another three (KU42.92A, KU44.11B and KU44.13A) are lgG2a Kappa isotype and one (KU42.73A) is IgM isotype (the latter not fully characterised). Immunofluorescence staining of tumour cells reveal membrane localization of the antigens recognised by the novel mAbs. In addition, treatment with mAbs KU42.77, KU42.92A, KU44.4C, KU44.22B and KU44.71A induced antigen downregulation and internalisation in BxPC-3 or AsPC-1 cancer cells. One of the mAbs, KU44.22B, demonstrated significant anti-tumour activity 'in vitro' on Capan-2 human pancreatic cancer cells inducing 94% growth inhibition at 300 nM with a IC50 of 4.5 nM. At concentrations up to 300 nM, the remaining mAbs did not significantly affect tumour cell proliferation. On the other hand, there was no evidence of reduced migration following treatment with any of the novel mAbs. Interestingly, mAb KU44.22B induced a significant increase in migration of both BxPC-3 and CFPAC-1 tumour cells, while mAbs KU44.2 and KU44.71A induced an increase in migration of BxPC-3 and CFPAC-1 cells, respectively. Immunoprecipitation and mass spectrometry analysis revealed the target antigens for the novel mAbs as follows: KU42.33C and KU43.12A (CD109 antigen), KU42.73A (lgμ chain C region), KU42.92A (Ag 243-5 protein precursor), KU44.13A (dipeptidyl peptidase 4), KU44.22B (integrin α3) and KU44.71A (integrin α6β4). KU42.77, KU44.2 and KU44.11B revealed matches with myosin-9, which could be attributed to cross-reactivity based on the significantly different pattern of antigen expression for those mAbs by flow cytometry. KU42.33C, KU42.92A and KU44.71A were the only mAbs that detected their target antigens by western blot. The identity of the target antigen of mAbs KU42.33C and KU43.13A (CD109 antigen) was confirmed using a commercially available anti-hCD109 mAbs for immunoprecipitation prior to antibody analysis via western blot. Finally, all the novel aAbs showed application as companion diagnostics for use in the immunohistochemical detection of target antigens, with most of them requiring antigen retrieval and signal amplification for visualization. In conclusion, the characterisation of the panel of novel mAbs developed in this study demonstrated their use as diagnostic and/or therapeutic tools in pancreatic cancer 'in vitro'. Further studies are warranted to fully explore their potential for therapy (e.g. as antibody conjugates to toxins, chemotherapeutic agents or radioisotopes) and diagnosis (e.g. immunohistochemistry of human cancer and normal tissue), as they might ultimately prove useful in the fight against pancreatic cancer.
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