Immunotherapy Projects

Immunotherapy Projects - Research at Peter Mac

Current Available Research Projects

Generation of a tripartite chimeric single chain receptor for enhancing cancer therapy.
(PhD or B.Sc. (Hons) project).
Supervisor: Dr. Phil Darcy

Adoptive immunotherapy involving genetic modification of T cells with TCRαβ genes or single-chain (scFv) chimeric receptors is emerging as a promising approach to specifically direct their activity toward tumour cells1,2. Recently we have shown that chimeric receptors comprising the co-stimulatory CD28 signaling domain linked in tandem with the CD3-ζ domain (scFv-CD28-ζ) could optimally trigger T cell function in vitro and in vivo3,4. Nevertheless, these gene-engineered T cells were less effective against established subcutaneous disease. A potential way to improve this approach is to increase their survival and persistence. Indeed several studies in patients have shown a good correlation between persistence of adoptively transferred T cells and their anti-tumour effects. Signaling molecules from the TNF receptor family that include OX-40, CD27 and 4-1BB, play an important role in both survival of T cells following activation and generation of an effective memory response. Hence, in this project, we propose to test whether incorporation of these various signaling molecules from the TNF receptor family into our existing chimeric receptor design (tripartite receptor) can enhance survival and anti-tumour activity of gene-modified T cells.

The project will involve a number of molecular and biochemical methods including DNA cloning, sequencing, flow cytometry, ELISA, cytokine and proliferation assays. The student will also become competent in tissue culture (retroviral transduction of T cells and tumour cells) and handling of mice. We are looking for a highly motivated student who is interested in developing effective treatments for cancer.

References
1 Morgan RA, Dudley ME, Wunderlich JR, et al. Science 2006;314:126-9.
2 Kershaw MH, Teng MW, Smyth MJ, Darcy PK. Nat Rev Immunol 2005;5:928-40.
3 Haynes NM, Trapani JA, Teng MW, et al. Blood 2002;100: 3155-63.
4 Haynes NM, Trapani JA, Teng MW, et al. J Immunol 2002;169:5780.

Investigating the differences between responding and non-responding tumours following immunotherapy.
(PhD or B.Sc. (Hons) project)
Supervisor: Assoc. Prof. Michael Kershaw

While testing various immunotherapies against cancer in mice, we have developed some combinations of therapeutic agents that can cause complete regression of established tumors in a large proportion of mice. One such combination involves three immune-stimulating antibodies specific for CD40, CD137 and DR5. This is very exciting and we continue to develop these approaches further in trying to optimize them for eventual clinical translation. However, it is also clear that a proportion of tumours do not respond very well to therapy and continue to grow and metastasize rapidly. This is despite the mice being from an inbred strain and the tumour cells used for injection being the same for all mice. Reasons for this observed variability in tumour responses in different mice may be as simple as “random experimental variation” or there may be a more definitive interesting biological reason which could help us identify limitations to therapies. Therefore, this project seeks to investigate why some tumours respond while others do not. The project essentially involves injecting tumours into mice and then taking biopsies for analysis while leaving the bulk of the tumour with the combined therapy. Tumours are then monitored for response to therapy and corresponding biopsies samples analyzed to determine differences in biological parameters. Anticipated techniques to be used in the investigation include gene expression analysis using DNA microarray and quantitative PCR. We will also look at the cellular makeup of tumours using various histological techniques. In this way we may derive insight into molecular and cellular events that may dictate whether tumours will respond to treatment. This may allow us to design appropriate therapies for individual patients and tumours.

Reference of interest: Uno et al. Eradication of established tumors in mice by a combination antibody-based therapy. Nature Medicine 2006, June 12 (6): 694-698.

Generating efficient anti-cancer lymphocytes using a recombinant combinatorial library.
(PhD or B.Sc. (Hons) project)
Supervisors: Assoc. Prof. Michael Kershaw and Dr. Phillip Darcy.

Approximately 1 in 3 Australians develop cancer at some stage in their lives, and despite advances in the major treatment options such as surgery, chemotherapy and radiotherapy, almost 50% of cancer patients die of their disease. Clearly, the development of other treatment options is a high priority. In this regard, immunotherapy offers great promise as a treatment for cancer, but the immune system is often tolerant of tumours. Indeed, effective lymphocytes specific for tumour antigens are often not present within the immune repertoire of patients. Nevertheless, genetic modification of T cells can be used to generate lymphocytes with the ability to recognize and respond against tumour cells. This process is termed genetic redirection. Gene constructs, that have been successfully used to redirect T cell function against tumour cells, are often chimeric and encode extracellular domains consisting of single-chain antibody (scFv) specific for tumour antigen linked to intracellular domains able to trigger T cell activation. Several gene formats have been compared by laboratories around the world for their ability to trigger T cells to engage and destroy tumour cells. The latest chimeric formats link scFv through hinge and transmembrane regions to several signalling domains including the zeta chain of the CD3 complex and the costimulatory domain from CD28. However, despite some success of genetically redirected T cells against tumour cells, the magnitude of the T cell response has been suboptimal and does not approach that possible against non-self antigens such as viral proteins. In order to enhance the activity of T cells against tumour cells, investigators are continuing to try different signalling domains in the cytoplasmic region of chimeric genes. However, previous methods involve selection of candidate molecules followed by genetic cloning and functional characterisation in T cells. Such methods are laborious and time consuming, which results in slow progress towards the generation of optimal tumour-specific T cells for the adoptive immunotherapy of cancer. To overcome the limitations of this methodology, the aim of the current research project is to construct a recombinant library of T cell signalling molecules and use it to genetically modify human T cells to endow them with enhanced anti-cancer function. This library approach should enable the rapid testing of many hundreds of different receptor formats to select the best for cancer immunotherapy. The project will involve: (1) The use of molecular biology to generate a DNA library; (2) The genetic modification of human T cells using this library and retroviral vector technology; (3) Comparing the functional responses of genetically modified T cell clones against tumour cells using cytotoxicity and cytokine secretion assays.

Reference of interest: MH Kershaw et al. Supernatural T cells: genetic modification of T cells for cancer therapy. Nature Reviews Immunology 2005 Dec;5(12):928-40.

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