The vision for our Centre has been to develop new and innovative medicines for poorly treated diseases through the discovery of new knowledge about the control of normal and abnormal cell homeostasis. This involves the characterisation of cell surface receptors, intracellular signal transduction pathways and regulatory mechanisms. Aberrations in cell to cell signalling through cell surface receptors, as well as mutations to intracellular signalling mechanisms,have long been recognised as targets for drug discovery.

New drugs present themselves in two basic forms: molecules of small molecular weight, with the potential for being orally active; and complex biological molecules of high molecular weight, referred to generically as 'biologicals'. Biologicals are of immense significance in meeting clinical needs due

to their ability to target very specific interactions and pathways, however, they are not amenable to oral administration. Combining these two approaches i.e. the oral activity, ease of handing and the relative ease of manufacture of small molecules and the high specificity along with the ability of large complex biological molecules to be able to function as ‘mimetics’ or ‘antogonists’, is a primary goal of many pharmaceutical R & D programs.

The Centre is well positioned to exploit this opportunity through its expertise in cell biology, animal models of disease, pre-clinical testing systems, protein chemistry, proteomics and, with the introduction of medicinal chemists last year, a strong small-molecule focused chemistry resource. Through WEHI, the CRC-CGF has recently had access to high-throughput screening in its discovery arsenal. This resource consists of targeted chemical libraries that are being made by our medicinal chemists and in the final year of funding there continues to be a concerted effort to identify new drugs targeting the biological molecules that have been discovered during the life of our Centre. In addition to this new capacity in the area of small molecule development, there are also great opportunities for the Centre to continue exploiting biologicals as drug candidates.

Many treatments for asthma today do not target the mechanisms that underlie disease progression, and in some cases are associated with significant side-effects and decreased efficacy after prolonged use. Despite the therapeutic advances made over the past 25 years, the prevalence and severity of asthma has risen substantially and there is clearly a need to develop new drugs against novel therapeutic targets. The commercial potential for a new and effective asthma medication is very significant with the current market size for asthma drugs estimated to be in excess of US$5 billion.

 IL-13Rα1 was discovered within the Centre (at WEHI) as part of a screen to identify novel cytokine receptors. The receptor is a functional component of both the IL-4 and IL-13 receptors. Recent published studies in mice have highlighted the role of IL-13 in the development of allergic asthma. Mice primed to develop asthma-like symptoms showed reduction or ablation of such symptoms when treated with a truncated form of IL-13Rα2 to block the effects of IL-13. Confirming the role of IL-13Rα in these pathologies, repeated administration of recombinant IL-13 to the airways of naive mice induced an asthma- like response.

These reports and a variety of other studies further support a central role for IL-13 in the development of mouse allergic airway disease and, by extension, human asthma. In humans, recent collaborative studies have demonstrated IL-13Rα1 expression in a variety of cells found in biopsies of human asthmatic lung. The data indicate that IL-13 plays an important role in the development of crucial features of airway disease. On this basis, components of the IL-13 signalling pathway, such as IL-13Rα1, represent novel therapeutic targets for asthma, particularly for large molecule-based drugs such as monoclonal antibodies.

This approach is being taken by the Centre as the main focus of our current efforts in the area are directed towards the development and preclinical characterisation of IL-13Rα1 specific therapeutic monoclonal antibodies (mAb) using a number of technology platforms. At the Amrad laboratories, humanisation of amouse IL-13Rα1 specific mAb (1D9), demonstrated to be a potent antagonist of IL-13 and IL-4 activity in both molecular and cell-based assays, has been completed. Stable CHO cell clones expressing the mAb have recently been generated and purification of mAb for further characterisation is in progress. A subset of the fully human mAbs generated using the Medarex HuMAb transgenic mouse technology have also been shown to be potent antagonists of IL-13 and IL-4 and are currently being characterized further.

The Centre's current efforts in this area are directed towards generating GM-CSFRα specific therapeutic monoclonal antibodies and we are using a number of different technology platforms to achieve this aim. Significant progress has been made with the humanisation of a mouse GM-CSFRα specific mAb, previously demonstrated to be a potent antagonist of GM-CSF activity. Following several framework changes to the 2B7-based CDR grafted Fab fragment, binding to the GM-CSFRα has been demonstrated. The collaboration with Cambridge Antibody Technology (CAT) to generate human mAbs against the GM-CSFRα, using CAT’s proprietary phage display technology, has also made very significant progress. Optimisation of a lead mAb has been completed and highly potent derivative mAbs have been identified.

To validate the LIF receptor and IL-11 receptor as targets for the development of novel contraceptive agents.

There is considerable evidence to suggest that IL-11 and LIF are essential for normal female fertility. Female mice lacking the gene for the IL-11 receptor or for LIF were found to be infertile. A close examination of these mice has revealed that LIF appears to be essential for implantation of the blastocyst into the uterine wall, while IL-11 is critical in the uterine decidualisation response that occurs immediately after blastocyst implantation. In human females, IL-11 also appears to be important for decidualisation and reduced levels of LIF expression have been correlated with infertility in some women.

With funding support from the CONRAD program, we are producing antagonists of the LIF receptor (LIFR) and IL-11 receptor (IL-11R) to test whether blockade of either of these signalling cascades is a valid strategy for the development of novel contraceptive agents. To do this we are generating engineered forms of the LIF and IL-11 cytokines that are able to act as antagonists. These antagonists work by competing with the wild type cytokinesfor binding to LIFR or IL-11R, but fail to engage the gp130 co-receptor required for productive signalling. We have now identified mutations in both LIF and IL-11 that prevent binding of the cytokine to gp130. When these mutants were recombinantly produced and tested on engineered cell lines that respond to either LIF or IL-11, they specifically inhibited thebiological activity of their respective cytokine.

To improve the potency of these antagonists, we are using phage display technology to identify additional mutations that enhance the affinity for binding to LIFR or IL-11R. Here the residues on the antagonist that are involved in receptor binding are randomly mutated and the resultant library of mutant proteins is displayed using phage technology. These libraries are then panned against the appropriate receptor protein in an iterative manner to select the highest affinity mutant from within the pool. To date, we have successfully applied this approach to the affinity maturation of the LIF antagonist. A number of mutations were identified which enhanced the affinity for binding to LIF receptor by approximately 1000-fold. This dramatic improvement in LIF receptor binding affinity resulted in a substantial enhancement of the antagonist activity of this protein. We are currently applying this same strategy to optimize the activity of the IL-11 antagonist. In a joint CONRAD-funded study with Associate Prof. Lois Salamonsen from Prince Henry’s Institute of Medical Research (Melbourne), we are now assessing the contraceptive potential of these antagonists in rodent animal models.

                        A model for ErbB2-heterodimer signalling.

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The information derived from the crystal structures described above has drastically altered the understanding of EGF receptor family interactions, pointing toward a new method of receptor activation and signalling. Using this information, key regions of the receptors that have been identified are currently being used in a strategy for developing novel inhibitors that specifically target these regions. Monoclonal antibodies have gained acceptance by the pharmaceutical industry as drugs with high levels of target specificity. The EGF receptor project team are currently using the defined regions of interest as targets for monoclonal antibody production and testing.

Similarly, through using in silico or ‘virtual’ techniques, the Centre is screening large structural databases for compounds that have the ability to fit into the molecular architecture of the two EGFR family members and appear to be essential for signalling. Any hits that are identified will be further screened using specially designed binding and cell culture assays to assess any inhibitory or indeed agonistic activity.

Project 3A - Soluble Truncated EGF Receptor

Cytokines and growth factors exert their biological effects by binding to specific cell surface receptors. Typically,after binding to their receptors,many cytokines induce receptor dimerisation and recruitment of JAK family kinases, which in turn leads to a series of intracellular molecular interactions culminating in transcription of target genes in the nucleus.

While much is understood about the positive regulation of cytokine signalling, until recently little was known about how these signalling cascades are attenuated. Like most important physiological processes, cytokine action needs to be controlled to ensure an appropriate response to the biological challenge without incurring extensive damage to host tissues. The discovery, within the Centre (at WEHI), of a family of proteins known as Suppressors Of Cytokine Signalling (SOCS) has revealed a class of proteins that are important mediators in the negative regulation of cytokine signalling.

The first protein in the family, SOCS-1, was discovered in 1997 and was shown to have a central SH2 domain but little or no homology to any other protein except CIS (cytokine-inducible SH2 protein). These two proteins share homology not only in the

Extensive analysis of SOCS-1 function in vivo has demonstrated that this molecule is a key regulator of interferon-gamma (IFNγ) signalling. In the absence of SOCS-1, excessive IFNγ signalling causes perinatal lethality due to liver degeneration and widespread inflammatory disease. Mice lacking both SOCS-1 and IFNγ survive to adulthood but display aberrant T cell activation and succumb to inflammatory lesions and polycystic kidney disease in the second year of life. Mice lacking SOCS-1 or both SOCS-1 and IFNγ show heightened resistance to viral infection compared with wild type mice. This suggests an important role for SOCS-1 in the regulation of Type I (IFNα and IFNβ) as well as Type II interferon (i.e. IFNγ) activity. Interestingly, recent data show that SOCS-1 deficient mice are more resistant to malaria infection, although the basis for this response has yet to be resolved. Together, these data suggest that SOCS-1 controls the balance between the beneficial effects of interferons and pathology induced by excessive host responses to these cytokines.

It follows then, that there is a potential beneficial role formodulation of SOCS-1 in infections or in IFN-treated diseases. IFNα is administered therapeutically to treat hepatitis and some cancers, while IFNβ is used to treat multiple sclerosis and IFNγ for osteoporosis and chronic granulomatous disease. A SOCS-1 antagonist may improve or even be able to replace these treatments.

Over the last 12 months great progress has been made towards the development of a high capacity assay that can be used to screen the WEHI/Bio21 Lead Discovery library for potential antagonists of the SOCS-2/GH receptor interaction. The SOCS-2 screening campaign is planned for the first quarter of 2003-2004, allowing the remainder of the year to complete the chemical and biological evaluation of the active compounds.

Immunofluorescent staining of cells transfected with STAT1 before (top left) and after (top right) cells

 were stimulated with LIF. Stat1 is shown in green and the nucleus is shown in blue.

To investigate the biology of Lyn tyrosine kinase and to identify any therapeutic applications that might arise from modulation of the enzyme with known or novel molecules. The past twelve months have been spent validating the Lyn protein tyrosine kinase as a drug discovery target. The Centre’s original data supported the concept that inhibitors of Lyn tyrosine kinase activity may be useful therapeutics for clinical applications such as the stimulation of haematopoiesis, the process by which blood cells are produced. However, while our studies have shown that Lyn-deficient mice have elevated numbers of myeloid colony forming cells, the mice are not advantaged following ablation of their haematopoietic system with chemotherapeutic drugs such as 5-fluorouracil. In addition, although Lyn-deficient mice showed enhanced erythropoiesis, they showed no difference in recovery of their red blood cell compartment with the anaemia-inducing drug phenylhydrazine.

Thus, these studies indicate that inhibitors of Lyn are unlikely to be useful therapeutics for the application of haematopoiesis. However, our current studies on the role of Lyn in innate immunity have indicated another potential application for Lyn inhibitors. Recent work in our laboratory has shown that Lyn is important in regulating an animal's innate immune response to bacterial products such as endotoxin. Future studies are aimed at initiating a drug discovery program based on Lyn kinase in collaboration with the High Throughput Chemical Screening and Medicinal Chemistry groups. A screen utilising the tyrosine kinase activity of Lyn is currently being developed and will form the basis of that which will be utilised in the high throughput library screening process as well as providing support for further development of any potential hits through the medicinal chemistry program.