No description available.

No description available.

No description available.

An in vitro model to mimic in vivo conditions was developed using adherent BM Mesenchymal Stromal Cells (MSCs), isolated from the iliac crest of normal donors. These "fibroblast-like" MSCs provide a supporting layer for haemopoietic differentiation. They lack expression of haemopoietic differentiation antigens and lineage markers:- CD34, CD45, HLA-DR, CD31 and CD68 but express Vimentin, alphaSmooth Muscle Actin and Ab-1 (fibroblast marker). The model was used to determine if MSCs could protect B-CLL B cells from spontaneous apoptosis and prevent the effects of the therapeutic monoclonal antibodies Rituximab (CD20) and Campath-1H (CD52), the proteasomal inhibitor MG132 and BisIX, a protein kinase C inhibitor. Levels of apoptosis were determined by flow cytometry, using Annexin V-FITC/PI. MSCs abrogated spontaneous apoptosis of B-CLL B cells in direct contact with them, over 4 days (80-90% viability, n=30). Rituximab and Campath-1H induced apoptosis in the B-CLL B cells that could not be prevented by contact with MSCs (n=6). MG132 and BisIX caused substantial apoptosis in B-CLL B cells (0-15% viability) but MSCs could protect against lower concentrations (200nM) of both drugs (70-90% viability, n=6). MSCs' protection of B-CLL B cells from spontaneous apoptosis was through the upregulation of the expression of Mcl-1 and Xiap, but not Survivin. The cell-cell interactions include the beta1, beta2, beta3 and beta4 integrins but have variable effects on B-CLL cases when bound by blocking antibodies. Therefore MSCs can maintain B-CLL viability in vitro and influence chemotherapeutic strategies. The model provides a reliable method to evaluate drug cytotoxicity and Mcl-1 offers a novel therapeutic target.

Amphipathic peptides with an a-helical structure disrupt membranes rich in negatively charged phospholipids and have antibiotic properties. They are toxic to eukaryotic cells if internalised by a suitable targeting mechanism. We have targeted one such peptide D(KLAKLAK)2 to haemopoetic cells by conjugating it to monoclonal antibodies which recognise lineage-specific cell-surface molecules. An anti-CD33: peptide conjugate was cytotoxic to one of three CD33-positive myeloid leukaemia lines, whereas an anti- CD 19: peptide conjugate efficiently killed three out of three B lymphoid lines with IC50 in the low nanomolar range. Cell lines which did not express the relevant antigen were resistant to the conjugates at the concentrations used. Peptide conjugated to anti-CD 19 and anti-CD33 antibodies had cytotoxic activity towards cells isolated from the peripheral blood of patients with chronic lymphocytic leukaemia (CLL) and acute myeloid leukaemia (AML) respectively. CLL cells isolated from heavily pre-treated patients or from poor risk category individuals, as determined by elevated expression of the ZAP-70 protein tyrosine kinase, were as sensitive to the conjugate as were untreated or good risk category patients. Cell death was shown to be by an apoptotic mechanism and independent of the level of expression of anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP. In addition the conjugate acted synergistically with chlorambucil. The data here suggest that anti-CD 19 D(KLAKLAK)2 displays a highly selective and potent cytotoxic activity against CLL cells and may be of therapeutic value in the treatment of this incurable leukaemia. We also panned a phage display library in an attempt to isolate a peptide that would bind to CD33 and thereby create a peptide with both targeting and cytotoxic activity towards malignant myeloid cells.

Dysregulation of the LM02 proto-oncogene as a result of reciprocal chromosomal translocation involving the T-cell receptor gene loci, is a hallmark of some cases of T-cell acute lymphoblastic leukaemia (T-ALLL an aggressive subset of leukaemia. Aberrant expression of the gene is also seen in a significant proportion of cases. LM02 activation following retroviral insertion is further directly implicated in the pathogenesis of T-ALL that occurred in two gene therapy trials, while T-cell overexpression of the gene can be used to model the disease in mice. These existing models have been developed within, towards the live imaging of tumours; and to study the effect of an additional genetic lesion, with the goal of reducing the latency to tumour development. The LM02 protein is a component of multi-protein DNA-binding complexes with a role in cell fate decisions. Protein-protein interactions between LM02 and partners, in particular TAll, are targets for drug development, though this is a challenging therapeutic area. Previous isolation of an intracellular antibody fragment able to bind i to LM02 and inhibit important biological functions and pre-clinical tumour formation, is an important advance. However, in the absence of robust platforms for the efficient delivery of such a macromolecule, it is argued that the optimum therapeutic strategy is to discover small molecules that can mimic the interaction with LM02. To this end, this study contributes additional information about the interaction surface of this scFv, and applies the antibody technology to study of the dynamic interactions between LM02 and its protein partners. Both would be greatly strengthened by a structural solution of the scFv-LM02 complex. Though further work is required in each of the contributing areas before the goal of anti-LM02 drugs is realised, ultimately the ability to target multiple cellular pathways is desirable, and an approach to studying this in model systems has been developed.

No description available.

The majority of patients with chronic myeloid leukaemia (CML) achieve a complete cytogenetic response (CCR) on imatinib. However, we cannot predict which patients will have a suboptimal response. We set out to investigate ways of predicting poor cytogenetic response to imatinib in patients with CML. We examined patients with CML for the possession of particular alleles of the rs2290573 and IL-I P +3953 polymorphisms because these polymorphisms had been linked with the rate of achievement of a major cytogenetic response (MCR). Following the discovery of a polymorphism (K247R) within the P-Ioop of BCR-ABL, we determined its frequency within both CML patients and healthy blood donors, and used in vitro biochemical and cellular assays to test its drug sensitivity. We examined patients with primary cytogenetic resistance to imatinib for the expression of genes associated with drug transport (hOCT 1, MDR 1, ABCG2, ABCCl, ABCA2, ABCC2, ABCC3, ABCC6 and MVP) and compared this to patients who achieved a complete cytogenetic response to imatinib. We used gene expression profiling of CML unselected white cells, and of CML CD34+ cells, to look for genes associated with poor cytogenetic response. We could not find a correlation between the possession of the rs2290573 and IL-I p +3953 polymorphisms, and rate of MCR in our patients. The K247R polymorphism was rare, but 3 out of 5 patients with the arginine allele failed to achieve a major MCR. Despite its position in the P-Ioop, in vitro assays showed K247R to have a drug sensitivity phenotype highly similar to wild type BCR-ABL. Imatini b non-responders had significantly lower pre imatinib gene expression levels of hOCT 1, and significantly higher levels of ABCC3. compared to responders. In addition, in imatinib non-responders we found that the expression level of a variety of drug transport genes changed with time on imatinib, but these changes did not reach statistical significance. Gene expression profiling of CML total white cells revealed that imatinib responders and non-responders had highly similar gene signatures, and that the noise created by different sample source, handling and cell phenotype limited the detection of changes in gene expression. We successfully developed a technique for selecting CD34+ cells from cryopreserved CML mononuclear cells, and preliminary analysis of the CD34+ cell microarray data does not identify any genes that are significantly differentially expressed between cytogenetic responders and non-responders. Possession of the rs2290573 and IL-βI+3953 polymorphisms did not aid prediction of achievement of MCR in our CML popUlation. In patients with CML, possession of the arginine allele of K247R should not be confused with the development of a kinase domain mutation, and the failure of 3 out of 5 patients to achieve a MCR is likely to be a chance finding in a small cohort, but further collection of response data on patients with K247R is required. Differences in drug transport gene expression may influence patients' responses to imatinib, by potentially causing inadequate intracellular imatinib concentrations. Gene expression profiling of CML unselected WBC does not allow prediction of response to imatinib, and work is ongoing to see if the technique proves more useful when using RNA derived from CML CD34+ cells.

Haematopoietic stem cell transplantation, one of the major treatments for Chronic Myeloid Leukaemia (CML), provides an allogeneic Graft-versus-Leukaemia (GvL) effect mediated by the donor T cells infused with the graft. The antigens recognized by these T cells are not yet identified. One candidate antigen is the CML specific BCR7ABL oncogene. The aim of this thesis is to investigate immunotherapeutic strategies for the treatment of CML targeted to BCR/ABL epitopes. We characterised BCR/ABL derived peptides that are naturally processed and presented on the surface of CML cells in the context of both HLA-A0301 and HLA-B0801 molecules. In order to screen for the presence of BCR/ABL specific T cells in CML patient samples, we generated HLA tetramer complexes, refolded with the appropriate BCR/ABL peptide. We detected the presence of low frequency (1%) BCR/ABL specific CDS+ T cells circulating in the peripheral blood of CML patients. The expansion of these BCR/ABL specific CTLs from healthy donors and CML patients was attempted using a number of different protocols, including peptide-pulsed dendritic cells. We generated leukaemia specific CTLs in some cases from healthy donors but not from patients. It was however possible in both groups to generate responses to viral antigens. We therefore investigated BCR/ABL immunogenicity, comparing CML patient responses to BCR/ABL antigens with other GvL associated tumour antigens. To circumvent a potential Antigen Presenting Cell (APC) deficiency in CML patients, we generated a standardized and unlimited source of artificial soluble Antigen-Presenting Complexes by cross-linking BCR-ABL HLA/peptide monomers with costimulatory molecules. These sAPCs successfully generated functional CDS BCR/ABL T cells from both healthy individuals and CML patients. The data presented here demonstrates the feasibility of BCR/ABL based adoptive T cell immunotherapy for CML patients, at least in the context of HLA-A0301.

(3aR)-14a-Acetoxy-3aß,4,14,14aa-tetrahydro-6,7,8-trimethoxybenzo[3,4]furo[3',4':6,7]cycloocta[1,2-f][1,3]benzodioxol-3(1H)-one (steganacin), 56, has been a popular synthetic target due to its perceived cytotoxic activity. Our proposed strategy for the construction of the key 8-membered ring embedded within steganacin was via an oxidative phenolic coupling of an appropriate (3R,4R)-4-(benzo[d][1,3]dioxol-5-ylmethyl)-3-benzyldihydrofuran-2(3H)-one analogue which were shown to be readily available from commercially available piperonal in six linear steps involving chain extension, reduction, trichloroacetylation and cyclisation via a copper-catalysed Atom Transfer Radical Cyclisation (ATRC) reaction. In this way, copper-catalysed ATRC reaction of (E)-6-(benzo[d][7’,9’]dioxol-1’-yl)allyl-1,1,1-trichloroacetate afforded (R)-4-{(R)-benzo[d][1,3]dioxol-5-ylchloromethyl}-3,3-dichlorodihydrofuran-2(3H)-one in good isolated yield as a mixture of diastereosiomers. Regiospecific functionalisation of these trihalides at the benzylic position (via an SN1, solvolysis, pathway), followed by dehalogenatioin and subsequent enolate alkylation afforded the key butyrolactone intermediates whose oxidative cyclisation was the key bond construct in our approach to steganacin. Contrary to our expectations it was observed that these substrates suffer intramolecular Friedel-Crafts alkylation reactions, favouring a 3a,4,9,9a-tetrahydronaphtho[2,3-c]furan-1(3H)-one (6 member ring) formation, rather than phenolic oxidative coupling reactions that would favour the steganacin-like (3aR,11aR,Z)-3a,4,11,11a-tetrahydrobenzo[4,5]cycloocta[1,2-c]furan-1(3H)-one (8 member ring) formation, when the oxidant has any Lewis acid capacity. Taking these observations into account we believe that by judicious choice of synthetic route the ATRC chemistry developed during the current research could be applied to a highly convergent (9 step) route to the synthesis of deoxypodophylotoxin.This work also describes, in detail, the efforts of this worker to establish and optimise a robust route that potentially can lead to the formation of steganacin via the alternative route of an initial microwave-assisted Pd-mediated biaryl coupling of either bromopiperonal or an halogenated derivative of the described γ-butyrolactones with an appropriate boronic acid derived from commercially available 3,4,5-trimethoxybenzalcohol to afford the biaryl scaffold present in steganacin. The completion of this synthesis was unfortunately left unaccomplished due to time constraints.