Investigation of monoclonal antibodies raised to human ovarian carcinoma cell lines

Monoclonal antibodies (MAb) were generated to multidrug resistant (MDR) and sensitive variants of the ovarian carcinoma cell line OAW42 which over-express the M D R associated protem LRP/MVP MAb 3/B6 was raised to the intrinsically resistant variant OAW42-SR Immunofluorescence and immunocytochemical analysis indicated that the antigen detected by MAb 3/B6 was expressed primarily on the external surface of the plasma membrane but was also found to be expressed in the cytoplasm of a series of human M D R cell lmes 3/B6 over-expression was associated primarily with cell lines which expressed the LRP/MVP. 3/B6 expression was studied in paraffin-embedded normal and malignant adult and in foetal tumour tissue. There was heterogeneous expression of the 3/B6 antigen and the LRP/MVP in normal adult and foetal kidney Low-level LRP/MVP expression was observed in 1/10 untreated malignant ovarian tumours while 3/B6 was absent. In two paired pre- and post-chemotherapy breast tumours sections, 3/B6 expression was observed in the post-chemotherapy sections only LRP/MVP expression was also observed in these sections. A new commercially available immunoprécipitation protocol based on biotm labelling of cellular proteins was extensively modified and improved for this project. The MAb 3/B6 was found to immunoprecipitate a 115 kDa un-glycosylated protein Immunoprécipitation experiments with anti-rat vault polyclonal serum, N2 and purified rat vault protems mdicated that MAb 3/B6 and LRP-56 (the standard MAb used to detect LRP/MVP) did not cross-react Competitive immunocytochemical studies confirmed these results Incubation of OAW42-SR cells with MAb 3/B6 did not have any effect on adnamycm drug accumulation or cellular proliferation. The antt-OAW42-SR MAb 5/C4 was also characterised by immunofluorescence and immunocytochemistry Results from these studies revealed that this MAb recognised a cytoplasmic antigen which migrated as 2 protein bands at 110 and 85 kDa by Western Blotting Further Western Blotting analysis indicated that MAb 5/C4 did not cross react with purified rat vault particles. The anti-OAW42-S MAb 3/E3 was partially characterised by immunofluorescence and immunocytochemistry. There did not appear to be any significant difference in expression of this antigen m a panel of multidrug resistant cell lines. It was not possible to determine the molecular weigh of the antigen by Western Blotting or immunoprécipitation. This suggested that the epitope was destroyed during sample preparation

Engineering mesothelin-binding proteins as targeted cancer diagnostics and therapeutics

Cancer is a significant global health concern; and traditional therapies, including chemotherapeutics, are often simultaneously toxic yet ineffective. There is a critical need to develop targeted cancer therapeutics which specifically inhibit molecules or molecular pathways essential for tumor growth and maintenance. Furthermore, a targeted therapy is only effective when a patient\u27s tumor expresses the molecular target; therefore, companion diagnostics, including molecular imaging agents, are a necessary counterpart of targeted therapies. Mesothelin (MSLN) is a cell surface protein overexpressed in numerous cancers, including triple-negative breast, pancreatic, ovarian, liver, and lung, with limited expression in normal tissues. Aberrant MSLN expression promotes tumor progression, metastasis, and therapeutic resistance, and is correlated with poor prognoses. Promising results from pre-clinical and clinical trials to target MSLN with antibodies, antibody derivatives, immunotoxins, and antibody-drug conjugates for therapy demonstrate the promise of MSLN-targeting methods; however, none are currently approved for routine clinical use, and limitations are emerging for targeting agents under development. New targeted diagnostic and therapeutic approaches for MSLN-positive tumors have potential for substantial impact in the clinic. In this work, I used yeast-surface display and directed evolution to engineer novel proteins based on the non-antibody fibronectin (Fn3) scaffold that bind to MSLN with high affinity and specificity. Soluble engineered proteins were expressed and purified to high yields from a bacterial system. In vitro cytotoxicity and apoptosis assays demonstrated the potential of the proteins as targeted cancer therapeutics for MSLN-expressing tumors, and the engineered proteins enhanced cancer cell sensitivity to chemotherapy. Towards the goal of using engineered Fn3 variants for targeted drug delivery applications, in collaboration with our collaborators, we synthesized and validated a novel protein-polymer conjugate drug delivery system. Finally, I established and validated appropriate in vivo tumor models to evaluate our engineered proteins as molecular imaging diagnostic agents. My work has provided a candidate set of engineered proteins that can be used as molecular targeted therapeutics, drug-delivery vehicles, and molecular imaging diagnostics for MSLN-positive tumors, towards the ultimate goal of providing targeted treatment and diagnostic options for cancer patients where no such treatments currently exist

Ca 125 in the physiology and pathology of the female reproductive tract: With particular reference to the diagnosis of ovarian cancer

This thesis addresses the hypothesis that "the CA 125 tumour associated antigen is of value in the diagnosis of ovarian cancer". The literature concerning CA 125 and diagnostic aspects of ovarian cancer are reviewed in chapter 1 and the research methodology and the characteristics of CA 125 assay systems are described in chapters 2 and 3. Chapters 4-6 describe studies of the compartmental distribution of CA 125 in the female reproductive tract in physiological and pathological states. The results suggest that CA 12 5 is a product of normal endometrium, pregnancy endometrium and benign ovarian tumours as well as malignant ovarian tumours, and that there is a physiological rise in serum CA 125 levels during menstruation and early pregnancy. It is concluded that the main factor influencing serum levels of CA 12 5 is the integrity of the blood: tissue barrier. Chapter 7 describes a prospective study to evaluate serum CA 125 measurement in the preoperative diagnosis of ovarian cancer. The results indicate that the accuracy of CA 125 measurement is superior to clinical criteria and similar to ultrasound. The highest diagnostic accuracy was achieved by combining CA 125, ultrasound and menopausal status in a risk of malignancy index. Chapter 8 describes phase 1 of a prospective study of screening for ovarian cancer amongst postmenopausal women. The results indicate that to achieve satisfactory specificity will require a multimodal approach combining CA 125 measurement with either pelvic examination or ultrasonography. Chapter 9 is a report of a phase 2 study of screening for ovarian cancer using the sequential combination of CA 125 and ultrasound. The preliminary results in 20,000 postmenopausal volunteers suggest that the lead time achieved over clinical presentation using this screening protocol is greater than 1 year. It is concluded that despite limitations of specificity and sensitivity, CA 125 is of value in the preoperative diagnosis of ovarian cancer and may have a role in a multimodal screening protocol for early stage disease

Protein Detection

The book explores distinct aspects of protein purification and characterization steps. It discusses solubility problems, resin selection tricks, and essential credentials in a purification process. In addition, the book examines aggregation and proteinopathy-related protein detection methods and reviews several essential protein detection and characterization methods in cancer for diagnosis, prognosis, and therapy, such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometry, western blot, mass spectrometry, and others