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Background: Discovery and validation of new antibody tractable targets is critical for the development of new antibody therapeutics to address unmet needs in oncology. Methods: A highly invasive clonal variant of the MDA-MB-435S cell line was used to generate monoclonal antibodies (MAbs), which were screened for anti-invasive activity against aggressive cancer cells in vitro. The molecular target of selected inhibitory MAb 9E1 was identified using immunoprecipitation/liquid chromatography-tandem mass spectrometry. The potential anti-tumour effects of MAb 9E1 were investigated in vitro together with immunohistochemical analysis of the 9E1 target antigen in normal and cancer tissues. Results: MAb 9E1 significantly decreases invasion in pancreatic, lung squamous and breast cancer cells and silencing of its target antigen, which was revealed as AnxA6, leads to markedly reduced invasive capacity of pancreatic and lung squamous cancer in vitro. IHC using MAb 9E1 revealed that AnxA6 exhibits a high prevalence of membrane immunoreactivity across aggressive tumour types with restricted expression observed in the majority of normal tissues. In pancreatic ductal adenocarcinoma, high AnxA6 IHC score correlated with the presence of tumour budding at the invasive front of tumours (P=0.082), the presence of perineural invasion (P=

Development of more effective therapeutic strategies for cancers of high unmet need requires the continued discovery of disease-specific protein targets for therapeutic antibody targeting. In order to identify novel proteins associated with cancer cell invasion/metastasis, we present here an alternative to antibody targeting of cell surface proteins with an established role in invasion; our functional antibody screening approach involves the isolation and selection of MAbs that are primarily screened for their ability to inhibit tumour invasion. A clonal population of the Mia PaCa-2, a pancreatic ductal adenocarcinoma (PDAC) cell line, which displays a highly invasive phenotype, was used to generate MAbs with the objective of identifying membrane targets directly involved in cancer invasion. Selected MAb 7B7 can significantly reduce invasion in a dose-responsive manner in Mia PaCa-2 clone 3 and DLKP-M squamous lung carcinoma cells. Using immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis, the target antigen of anti-invasive antibody, 7B7, was determined to be the heterodimeric Ku antigen, Ku70/80, a core protein composed of the Ku70 and Ku80 subunits which is involved in non-homologous end-joining (NHEJ) DNA repair. RNA interference-mediated knockdown of Ku70 and Ku80 resulted in a marked decrease in the invasive capacity of Mia PaCa-2 clone 3 and DLKP-M cells, indicating that Ku70/Ku80 is functionally involved in pancreatic and lung cancer invasion. Immunohistochemical analysis demonstrated Ku70/Ku80 immunoreactivity in 37 PDAC tumours, indicating that this heterodimer is highly expressed in this aggressive cancer type. This study demonstrates that a functional MAb screening approach coupled with immunoprecipitation/proteomic analyses can be successfully applied to identify functional anti-invasive MAbs and potential novel targets for therapeutic antibody targeting.

N -(3-ferrocenyl-2-naphthoyl) dipeptide esters ( 5–7 ) and N -(6-ferrocenyl-2-naphthoyl) dipeptide esters ( 8–10 ) were prepared by coupling either 3-ferrocenylnaphthalene-2-carboxylic acid 2 or 6-ferrocenylnaphthalene-2-carboxylic acid 4 to the dipeptide ethyl esters GlyAla(OEt) ( 5, 8 ), AlaGly(OEt) ( 6, 9 ), and AlaAla(OEt) ( 7, 10 ) using the standard N -(3-dimethylaminopropyl)- N ′-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt) protocol. All the compounds were fully characterized using a combination of 1 H NMR, 13 C NMR, DEPT-135 and 1 H- 13 C COSY (HMQC) spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and cyclic voltammetry (CV). In vitro , the cytotoxic effects of compounds 5–10 show improvements over the corresponding N -(ferrocenyl)benzoyl derivatives, with IC 50 values against the H1299 lung cancer cells ranging from 1.2 μM to 8.0 μM. N -(6-ferrocenyl-2-naphthoyl)-glycine- l -alanine ethyl ester 8 was found to be the most active derivative of the naphthoyl series so far, displaying an IC 50 value of 1.3 ± 0.1 μM. This value is slightly lower than that found for the clinically employed anti-cancer drug cisplatin (IC 50 = 1.5 ± 0.1 μM against H1299).

N - ortho, meta and para -(ferrocenyl)benzoyl dipeptide esters 2–10 were prepared by coupling ferrocenyl benzoic acids 1 (ortho, meta and para) to the dipeptide ethyl esters GlyAbu(OEt) 2–4 , GlyNva(OEt) 5–7 and GlyNle(OEt) 8–10 in the presence of N -(3-dimethylaminopropyl)- N′ -ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole. The compounds were fully characterized by a range of NMR spectroscopic techniques, mass spectrometry and cyclic voltammetry. The cytotoxicity of 3, 6 and 9 versus H1299 lung cancer cells were 10.5 μM, 19.1 μM and 18.9 μM, respectively, whereas N -{ meta -(ferrocenyl)-benzoyl}-glycine- l -alanine ethyl ester 11 and N -{ para -(ferrocenyl)-benzoyl}-glycine- l -alanine ethyl ester 12 gave IC 50 values of 4.0 and 6.6 μM, respectively. Therefore, an increase in alkyl chain length of the second amino acid also increases the IC 50 values. Cell cycle analysis of N -{ ortho -(ferrocenyl)-benzoyl}-glycine- l -alanine ethyl ester 13 suggests a block in the G2/M phase of the cell cycle.

N-ortho-ferrocenyl benzoyl dipeptide esters 2–6 were prepared by coupling ortho-ferrocenyl benzoic acid 1 to the dipeptide ethyl esters GlyGly(OEt) (2), GlyAla(OEt) (3), GlyPhe(OEt) (4), AlaGly(OEt) (5) and AlaPhe(OEt) (6). The compounds were fully characterized by a range of NMR spectroscopic techniques, mass spectrometry and cyclic voltammetry. The cytotoxicity of 3 and 5 towards lung cancer cells has been determined.

The interrogation of highly diverse repertoires of heterogeneous cell populations on a single cell basis increases the likelihood that a cell with unique characteristics will be identified. We have developed a new single cell analysis system comprising millions of bundled subnanoliter volume bioincubation chambers for the identification and recovery of target specific antibody secreting cells (ASCs). This platform integrates dual surface screening with dedicated user driven data analysis and automated cell recovery enabling multiple biophysical parameters to be tracked for millions of antibody leads in parallel. This direct clone analysis and selection technology is a clear deviation from current microfabricated well-based approaches and offers drastically enhanced screening throughput, simultaneous dual surface analysis, and rapid automated single cell recovery. The technology is also applicable to screening both bacterial and mammalian antibody secreting cells. We demonstrate the implementation and feasibility of this platform in identifying target specific antibodies from bacterial, hybridoma, and B cell libraries.

A series of N-(ferrocenyl)naphthoyl amino acid esters 5-18 has been prepared by coupling ferrocenyl naphthoic acids 3-4 to alpha-amino acids and linear amino acids in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt). The compounds were fully characterised by a range of NMR spectroscopic techniques, UV-Vis spectroscopy, mass spectrometry and cyclic voltammetry. X-ray crystallographic studies of the intermediate compounds 1-2 were also performed. Biological evaluation of the intermediates 1-2 and N-(ferrocenyl)naphthoyl amino acid esters 5-18 was performed in the H1299 non-small cell lung cancer (NSCLC) cell line and the Sk-Mel-28 metastatic melanoma cell line. The intermediates 1-2 failed to produce an effect in either cell line. Compounds 5-18 exhibited a strong anti-proliferative effect in the H1299 cell line, whilst the Sk-Mel-28 cells were slightly more resistant to these compounds. N-(6-ferrocenyl-2-naphthoyl)-gamma-aminobutyric acid ethyl ester 17 shows a particularly high activity in both the H1299 cell line (IC(50) = 0.62 +/- 0.07 microM) and the Sk-Mel-28 cell line (IC(50) = 1.41 +/- 0.04 microM).

Background A number of recent studies have suggested that cancer incidence rates may be lower in patients receiving statin treatment for hypercholesterolemia. We examined the effects of statin drugs on in vitro proliferation, migration and invasion of melanoma cells. Methods The ability of lovastatin, mevastatin and simvastatin to inhibit the melanoma cell proliferation was examined using cytotoxicity and apoptosis assays. Effects on cell migration and invasion were assessed using transwell invasion and migration chambers. Hypothesis testing was performed using 1-way ANOVA, and Student's t-test. Results Lovastatin, mevastatin and simvastatin inhibited the growth, cell migration and invasion of HT144, M14 and SK-MEL-28 melanoma cells. The concentrations required to inhibit proliferation of melanoma cells (0.8–2.1 μM) have previously been achieved in a phase I clinical trial of lovastatin in patients with solid tumours, (45 mg/kg/day resulted in peak plasma concentrations of approximately 3.9 μM). Conclusion Our results suggest that statin treatment is unlikely to prevent melanoma development at standard doses. However, higher doses of statins may have a role to play in adjuvant therapy by inhibiting growth and invasion of melanoma cells.

Monoclonal antibodies (MAbs) have emerged as an important therapeutic modality for the treatment of cancer, due to their high specificity, low toxicity, and the ability to activate components of the immune system. The research carried out in this thesis aims to identify novel antigens associated with cancer invasion, that could form the basis of anti-invasive therapeutic targets, through the generation of MAbs directed against the highly invasive MiaPaCa-2 clone 3 pancreatic cell line, and the MDA-MB-435-SF breast cancer cell line. Two MAbs were identified that could successfully block cancer invasion in vitro. MAb 7B7 G5 (2) significantly reduced invasion in the MiaPaCa-2 clone 3 pancreatic cell line; the SKBR-3 and MDA-MB-231 breast cancer cell lines; the DLKP-M and H1299 lung cancer cell lines; the SNB-19 glioma cell line and the HCT-116 colon cancer cell line. Inhibition of invasion was also observed in the Lox IMVI melanoma cell line, but not significantly so. This MAb also significantly decreased cell motility in the MiaPaCa-2 clone 3 cell line. MAb 9E1 24 (6) significantly decreased cell invasion in the MiaPaCa-2 clone 3, MDA-MB-231, DLKP-I, DLKP-M, H1299, C/68 and Lox IMVI cell lines. Invasion was also inhibited in the SKBR-3 cell line, but not significantly so. Surprisingly, invasion was increased in the HCT-116 colon cancer cell line, following incubation with this MAb. Other invasion-related processes were also decreased following incubation with the MAb 9E1 24 (6) and 7B7 G5 (2); MiaPaCa-2 clone 3 adhesion to fibronectin, and MMP-9 activity in the MDA-MB-231 breast cancer cell line. Immunohistochemical analysis of 9E1 24 (6) revealed that its target antigen is expressed to varying degrees in a wide range of tumour types (colon adenocarcinoma, pancreatic, breast, B-Cell lymphoma, Retinoblastoma and Glioma). Weaker staining was observed in normal colon, liver and prostate tissues. MAb 9E1 24 (6) was shown to react with a 75kDa protein band on Western blot analysis. Immunoprecipitation studies, followed by LC-MS/MS analysis, revealed that its target antigen was Annexin A6, a 75kDa cellular calcium and phospholipid binding protein. This was further corroborated by decreased expression of the reactive 9E1 24 (6) band in Annexin A6-silenced cells. siRNA silencing of Annexin A6 significantly reduced invasion in the MiaPaCa-2 clone 3 and DLKP-M cell lines, suggesting a role for this protein in the invasion process. A cross-linked immunoprecipitation approach with MAb 7B7 G5(2) revealed two bands at approximately 70 and 80kDa. LC-MS/MS analysis identified these as Ku70 and Ku80 respectively, two subunits of the Ku heterodimer, which is involved in DNA double strand break repair. siRNA silencing of these two subunits in the MiaPaCa-2 clone 3 and DLKP-M cell lines significantly reduced levels of invasion and motility, indicating that they play a role in both processes. Immunofluorescence analysis on Ku70 and Ku80 silenced MiaPaCa-2 clone 3 cells revealed a significant decrease in MAb 7B7 G5 (2) reactivity on Ku80, but not Ku70, silenced cells, suggesting that the Ku80 subunit is the main target antigen for MAb 7B7 G5 (2). The research presented in this thesis is proof of principle of how MAbs can be successfully generated that specifically target invasion-related proteins, and can block cancer invasion in vitro. The identified proteins may have the potential to become useful therapeutic targets for the treatment of invasive cancers, and could lead to the development of new drugs that specifically target metastatic cancer cells.