Project Details

Colorectal cancer is one of the most characterized tumour types in term of the underlying genetic aberrations and can be broadly separated into two major molecular subtypes: diploid tumours with microsatellite instability (MIN) and aneuploid tumours with chromosomal instability (CIN). While the genetic mutations that leads to the microsatellite instable phenotype is well characterized, the cause of chromosomal instability (CIN) phenotype is relatively unknown. We have recently demonstrated that a novel class of targeted therapeutics known as histone deacetylase inhibitors (HDACi), preferentially induce G2-M arrest in tumour cell lines that exhibit the MIN, compared to the CIN phenotype. The goal of this Honours project is to investigate potential mechanisms by which CIN tumours escape activation of the mitotic checkpoint in response to HDACi treatment, by comparing the gene expression changes induced by HDACi in MIN vs CIN tumour cell lines. Findings from this study are designed to enhance our understanding of the mechanism of action of this novel class of cancer therapeutics.

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Project Details

Colon cancer is one of the leading causes of cancer related deaths in Australia, and new treatment strategies for this disease are urgently needed. Histone deacetylase inhibitors (HDACi) are an emerging class of cancer therapeutics that induce apoptosis of colon cancer cells through epigenetic mechanisms. We have previously identified colon cancer cell lines that are highly sensitive and resistant to HDAC inhibitors. The goal of this Honours project is to identify molecular markers which can predict sensitivity to HDAC inhibitors, by comparing the epigenome of colon tumours sensitive and resistant to this drug. Findings from this study may help guide future clinical trials of this drug by identifying colon cancer patients likely to respond to this treatment.

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Background: The development of recombinant antibodies for cancer therapy has emerged as one of the most promising areas in oncology therapeutics. The approval of Herceptin for the treatment of breast cancer as well as the approvals of Erbitux and Avastin for colon, head and neck, and renal cell cancer, confirm the impact of antibody therapeutics in oncology. In addition, a further five antibodies have been approved by the FDA for cancer indications, including Rituximab, Campath, Myelotarg, Bexxar, and Zevalin, the latter three involving payload delivery by antibodies (isotopes and toxins). In view of the potential of this approach for cancer therapy, antibody based therapeutics are of major importance and improvements in antibody efficacy have immediate relevance for new cancer therapeutics.

The Lewis Y (LeY) antigen is a type 2 blood group related difucosylated oligosaccharide. The LeY antigen has been shown to be expressed by 60 - 90% of human carcinomas of epithelial cell origin. Our group in the Ludwig Institute for Cancer Research (LICR) has developed a humanised IgG1 anti-LeY monoclonal antibody (mAb) hu3S193. Hu3S193 has potent immune effector function, including complement-dependent cytotoxicity (CDC) (IC50 1.0ug/ml) and antibody-dependent cellular-cytotoxicity (ADCC) (IC50 0.5ug/ml) against LeY expressing tumour cells, and has minimal difference in affinity or effector function compared to the parent murine antibody 3S193. Hu3S193 has also been shown to have significant anti-tumour effect in animal models. We have also successfully labelled hu3S193 with Indium-111 (111-In), with retention of binding affinity, and demonstrated successful targeting of Ley expressing xenografts in nude mouse models. We completed a first-in-man Phase I trial of hu3S193 in patients with LeY expressing tumour demonstrating safety and excellent tumour targeting. A key finding from our clinical studies of hu3S193 was the long half-life in-vivo of the hu3S193 IgG1, which while optimal for therapeutic immune effector function, restricts the ability to use hu3S193 as a diagnostic, or for therapeutic payload delivery to cancer cells (eg isotopes/toxins). The pharmacokinetics of intact IgG is known to be strongly influenced by Fc binding to FcRn (the neonatal receptor). The LeY research project involves the design and engineering of novel humanised IgG constructs with improved pharmacokinetics for optimal payload delivery.

Honours project: Six hu3S193 mutants will be produced by transient expression in Freestyle 293 cells. Mutant antibodies will be affinity purified. Quality control assessments and initial in vitro characterization of the mutants will include SDS-PAGE analysis of 293 cell supernatants and purified antibodies, HPLC (High Performance Liquid Chromatography), BiaCore and FACS (Fluorescence Activating Cell Sorting) analysis with LeY-positive cancer cells. In vivo biodistribution studies in xenograft models will explore pharmacokinetics and tumour uptake of radiolabelled mutant antibodies compared to the parental hu3S193. Whole body imaging through SPECT (Single Photon Emission Computed Tomography)/CT (Computed Tomography) analysis will be used to select the mutants with highest tumour to background ratios and optimal half-live (t1/2) and area under the curve (AUC) values.

Key techniques: molecular biology, site-directed mutagenesis, FACS, ELISA, transient transfection, protein production, protein purification, radiolabelling, tissue culture, xenograft models, SPECT/CT imaging

Further Information: ingrid.burvenich@ludwig.edu.au

References

1.Scott, A.M., et al., Construction, production, and characterization of humanized anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solid tumors. Cancer Res, 2000. 60(12): p. 3254-61.

2.Clarke, K., et al., In vivo biodistribution of a humanized anti-Lewis Y monoclonal antibody (hu3S193) in MCF-7 xenografted BALB/c nude mice. Cancer Res, 2000. 60(17): p. 4804-11.

3.Clarke, K., et al., Therapeutic efficacy of anti-Lewis(y) humanized 3S193 radioimmunotherapy in a breast cancer model: enhanced activity when combined with taxol chemotherapy. Clin Cancer Res, 2000. 6(9): p. 3621-8.

4.Scott, A.M., et al., A phase I biodistribution and pharmacokinetic trial of humanized monoclonal antibody Hu3s193 in patients with advanced epithelial cancers that express the Lewis-Y antigen. Clin Cancer Res, 2007. 13(11): p. 3286-92.

5.Ramsland, P.A., et al., Structural convergence of antibody binding of carbohydrate determinants in Lewis Y tumor antigens. J Mol Biol, 2004. 340(4): p. 809-18.

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