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Title: Development and characterisation of novel monoclonal antibodies against colorectal tumour cells for use in cancer diagnosis and therapy
Author: Ali, Sumaira
Awarding Body: Kingston University
Current Institution: Kingston University
Date of Award: 2013
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Colorectal cancer is a major public health problem and a leading cuase of cancer deaths in the western world. At present, three monoclonal antibodies (mAbs) namely anti-epidermal growth factor receptor (EGFR) cetuximab and panitumumab and anti-vascular endothelial growth factor mAb bevacizumab have been approved for the treatment of patients with metastatic colorectal cancer. However, many patients simply do not respond, or the cancer aquires resistance following a short course of therapy. The aim of this study was to develop a new panel of mouse monoclonal antibodies directed against other cell surface antigens which are over-exoressed on human colorectal tumour cells using hybridoma technology and to investigate their potential as diagnostic and therapetic agents. A panel of human colorectal cancer cell lines, established from colorectal cancer patients at different stages of the disease, were employed as the source of immunogen for immunisation of a group of mice. Seven novel mouse hybridomas were developed, secreting seven novel mAbs named KU2.77, KU2.90, KU3.39, KU3.47, KU3.61, KU4.76 and KU4.94. Following purification, the binding efficacy of each purified mAb to a large panel of colorectal tumor cells, human foreskin fibroblast (DE532) and human immortalized keratinocyte (HACAT) cells was determined using ELISA and flow cytometry. All seven mAbs are directed against cell surface antigens which are over-expressed on human colorectal cancer cells. The differential binding of these mAbs to the various human colorectal tumour cells and to the other cells suggests that these antibodies may be directed against at least three distinct over-expressed cell surface antigens. Interestingly, of the seven mAbs examined, KU3.47 and KU4.94 did not bind to DE532 and HACAT cells suggesting that the antigen recognised by these two antibidies may be tumour specific antigens. at 300nM, with the exception of mAb KU3.39 which induced slight growth inhibition of CCL-228 and Colo2 cells, the growth of other colorectal cancer cells were not inhibited by the any of the antibodies while only two mAbs (KU2.77 and KU2.90) inhibited the migration of CCL-228 cells. Out of the seven novel mAbs, KU3.47, KU4.94 and KU3.61 stained their respective antigens in formalin-fixed, paraffin-embedded, colorectal tumour cell pellets, suggesting that these antibodies are directed against a sequential determinant on the antigen and could therefore have potential for use as tools for investigating the expression pattern, prognostic significance and predictive value of such antigens in cancer patients. The preliminary results of immunoprecipitation followed by mass spectroscropy revealed that three newly developed mAbsKU2.77, KU3.39 and KU4.94 may recognise or cross react with integrin alpha-3, Large neutral amino acid transporter small subunit1 (LAT-1) and apo-lipoprotein A-IV respectively. Since all seven novel mAbs are directed against over-expressed cell surface antigens, they could form excellent tools for use in basic research for investigating the role of such antigens in the progression of human cancers. Further investigations are warranted to identify the target antigens recognised by each of the novel antibodies and to determine their full potential as diagnostic agents and therapeutic agents when conjugated to various toxins, drugs or radioisotopes.
Supervisor: Modjtahedi, Helmout ; Khan, Gulfraz ; Jones, Lucy ; Dalgleish, Angus Sponsor: Higher Education Commission of Pakistan ; University of Karachi
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Cancer studies