Hematopoietic growth factor mimetics: From concept to clinic

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Cytokine & Growth Factor Reviews 20 (2009) 87–94 www.elsevier.com/locate/cytogfr

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Hematopoietic growth factor mimetics: From concept to clinic Michelle Perugini a,b,*, Antiopi Varelias c, Timothy Sadlon b,d, Richard J. D’Andrea a,b,d,e,* a

The Division of Haematology and The Centre for Cancer Biology, Hanson Institute and SA Pathology, Adelaide, South Australia 5000, Australia b Women’s and Children’s Health Research Institute, North Adelaide, South Australia, Australia c Queensland Institute of Medical Research, Brisbane, Queensland, Australia d The Centre for Stem Cell Research and the Schools of Paediatrics and Reproductive Health, and Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia e The Division of Haematology and Oncology, The Queen Elizabeth Hospital, Woodville, South Australia, Australia Available online 14 February 2009

Abstract Hematopoietic growth factor (HGF) mimetics offer a number of attractive advantages as therapeutic agents. Small chemical compounds, in particular, provide reduced cost and oral availability. As many of these mimetics are unrelated in structure to the normal cytokine the immunogenic response is not a significant issue. Isolation of small peptide agonists for erythropoietin (EPO) and thrombopoietin (TPO) receptors has been associated with significant translational challenges and here we summarize approaches used to achieve the potency and stability required for clinical utility. We also compare and contrast the initial screening approaches, and the translational and clinical issues associated with two recently approved TPO mimetics, romiplostim and the orally available eltrombopag. Finally we summarize the development and clinical findings for the EPO mimetic, HematideTM, consider alternative approaches, and discuss the future potential for isolation of growth factor (GF) mimetics. Crown Copyright # 2009 Published by Elsevier Ltd. All rights reserved. Keywords: Growth factor; Mimetic; Thrombopoietin; Erythropoietin; Hematopoiesis

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation of hematopoietic growth factor mimetics . Thrombopoietic agents . . . . . . . . . . . . . . . . . . . . Erythropoietin mimetics . . . . . . . . . . . . . . . . . . . Development of further cytokine mimetics . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction * Corresponding authors at: The Division of Haematology and The Centre for Cancer Biology, Hanson Institute and SA Pathology, PO Box 14, Rundle Mall, Adelaide, South Australia 5000, Australia. Tel.: +61 8 8222 3639; fax: +61 8 8222 3139. E-mail addresses: [email protected] (M. Perugini), [email protected] (R.J. D’Andrea).

Hematopoietic growth factors (HGFs) control the proliferation and differentiation of blood stem and progenitor cells and modulate effector functions in mature cells of the immune system. The advent of recombinant forms of these growth factors has provided therapies for augmenting or

1359-6101/$ – see front matter. Crown Copyright # 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cytogfr.2009.01.002

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manipulating blood cell production in a number of settings and some key HGFs are now in extensive use in the clinic. Recombinant-human erythropoietin (rHuEPO) is one of the most widely used HGF’s in the clinic and has already exceeded US$ 29 billion in sales [1]. Although in widespread use, rHuEpo therapy is associated with significant expense (approximated at >$10,000/year) associated with the requirement for repeated administration [1]. EPO is essential for the maintenance of red blood cell production and defects in EPO production result in anemia which is associated with a variety of symptoms including fatigue, impaired mental function and respiratory distress. In humans the major site of EPO production is the kidney and, as such, anemia is a major secondary complication arising from chronic kidney disease (CKD) [2]. Anemia is now also recognized as a serious secondary factor in a number of other clinical settings including HIV/AIDS [3], some forms of cancer [4–6], inflammatory bowel disease [7] or as a consequence of cancer therapy [8,9]. Importantly, treatment with rHuEPO significantly ameliorates the symptoms of anemia [8,10] providing a safe and effective therapy that leads to improvements in disease indices, patient survival and quality of life [9]. rHuEPO exists in the form of darbepoetin alfa (Aranesp1, Amgen), epoetin alfa (Eprex1, Janssen-Cilag), epoetin beta (NeoRecormon1, Roche) and CERA (continuous erythropoietin receptor activator; Mircera) and is now the most extensively used HGF in the clinical setting [11,12]. Another key HGF is granulocyte colony stimulating factor (G-CSF) which has a major role in controlling the proliferation and differentiation of cells of the granulocyte lineage and has proven to be a safe and effective treatment for severe neutropenia (defined as absolute neutrophil counts below 0.5  109 L 1) which leaves the patient at risk of severe and life-threatening bacterial and fungal infections. rHuG-CSF exists in the form of NEUPOGEN1 (r-metHu GCSF Filgrastim, Amgen), Granocyte1 (rHuG-CSF Lenograstim, Aventis Pharma) and Neulasta1 (pegylated G-CSF Pegfilgrastim, Amgen) and is used to alleviate neutropenia in chemotherapy and hematopoietic stem cell transplantation recipients [13,14] as well as in the majority of severe chronic neutropenia (SCN) cases. It has also been used routinely to mobilize peripheral blood stem cells, a major source of stem cells used in autologous and allogeneic transplantation [15]. Whilst the established clinical use of these recombinant HGFs is a success story for genetic engineering, treatment is associated with high cost and potential immunogenic reaction. The recombinant proteins have short plasma half-life (4–13 h) necessitating repeated parenteral administration associated with frequent clinical visits and problems with patient compliance in some chronic disease states [10]. This route of administration can be particularly distressing for children [16]. The half-life of these proteins in the circulation has been extended by protein modification, for example asparagine(N)-linked glycosylation of rHuEPO

(Aranesp) has successfully increased half-life by 2-fold to 3fold allowing once a week dosing [1,17] and PEGylation of rHuEPO (CERA) has prolonged survival in the circulation to 135 h [11] allowing once a month administration [18]. An alternative approach, successfully used for growth hormone, involved fusion of the growth factor to its receptor extracellular domain [19]. In rare cases administration of these recombinant HGFs is associated with a detrimental immunogenic reaction [20,21]. A systemic response to the HGF leads to a reduction in the level of the endogenous HGFs and a negative clinical response. This was a significant problem when recombinant forms of thrombopoietin (TPO) were developed (see below). In this case the recombinant HGF was withdrawn from trials and a number of alternative approaches have been used to identify agonists of the receptor for TPO. These approaches have shown that it is possible to identify small-molecule drugs suitable for oral administration providing simple and effective therapy and overcoming several of these issues specific to the recombinant HGF. In this review we will briefly discuss the approaches used to generate HGF mimetics, the current clinical status and specific advantages and issues associated with each. As there is significant potential for the use of mimetics for other GFs these will be discussed briefly as well.

2. Isolation of hematopoietic growth factor mimetics Isolation of mimetics has occurred through a number of approaches. These have been greatly facilitated by the cloning of receptor subunits for a number of cytokine receptors in the early-1990s which allowed large-scale screening approaches based on receptor binding or function. Large combinatorial phage display libraries of 109 phage or more have been screened to identify binders by affinity purification. Peptides with bioactivity have been identified from these as lead molecules for further development. The generation of specific cell-based assays for receptor activation has been particularly important for large drug/ chemical library screens. The requirement for sophisticated chemistry, access to large complex chemical libraries and high-throughput automation technology has meant that large pharmaceutical companies have performed the majority of these screens. These approaches have been most successful in generating mimetics for TPO and EPO which are now in clinical use and we will focus on these, using them to illustrate the advantages and drawbacks of each approach. Table 1 lists new agents with thrombopoietic and erythropoietic activity that have been approved by the U.S. FDA or are in varying stages of clinical trials.

3. Thrombopoietic agents The biggest clinical need for an alternative to the recombinant HGF is the treatment of reduced platelet counts

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Table 1 New agents with thrombopoietic and erythropoietic activity. Trade name

Alternative name(s)

Mimetic activity

Structure

Company

Clinical trial stage

FDA approval

Approved treatment

Romiplostim Eltrombopag

TPO TPO

60 kDa peptibody 442 Da small molecule

Amgen GlaxoSmith Kline

Phase III completed Phase III completed

Yes (2008) Yes (2008)

ITP ITP

HematideTM

AMG-531/NPlate SB497115/ Promacta#/RevoladeTM AF37702

EPO

No

N/A

CERA

EPO

Affymax Inc., Takeda Pharmaceutical Ltd. Roche

Phase II/III current

Mircera

Phase II completed, Phase III current

Yes (2007)

Renal Anemia

ABT007

Fab-EPOR

EPO

PEGylated synthetic peptide rHuEpo 60 kDa methoxypolyethylene glycol polymer Humanized EPOR Antibody

Preclinical trials

No

N/A

associated with immune thrombocytopenic purpura (ITP) or as a result of cancer chemotherapy. Chronic ITP is characterized by autoantibody-induced platelet destruction and reduced platelet production. Following the cloning of TPO a number of initial recombinant thrombopoietic agents were generated by pharmaceutical companies. These included rHuTPO (Amgen), PEG-rHuMGDF (Amgen) which comprised the N-terminal 163 residues of TPO attached to polyethylene glycol, and a recombinant TPO fusion protein (Promegapoietin; TPO/IL3 fusion protein, Searle R&D) (discussed in Refs. [22–24]). Despite initial success in clinical trials for chemotherapy and chronic ITP patients these agents were discontinued following immunological reaction to rHuMGDF in a small number of healthy volunteers (13 of 535) and associated drops in platelet counts (reviewed in Kuter et al. [25]). In two cases pancytopenia developed [26,27] and at least 4 of 650 chemotherapy patients, treated with rHuMGDF were also shown to have developed auto-antibodies. This neutralizing response did not occur with rHuTPO possibly due to the fact that it was administered intravenously rather than subcutaneously like rHuMGDF [28]. The development of second-generation thrombopoietic agents began with the isolation of peptide mimetics of TPO by Cwirla et al. [29]. These hormone binding site (HBS) peptides were isolated by Affymax, Inc., using affinity purification from phage display libraries with the recombinant receptor. The active peptide displays no homology with native TPO and whilst potency of the monomeric peptide was very low this was dramatically improved following chemical dimerization, which generated a molecule with potency equivalent to rHuTPO [29]. This molecule has been further developed using PEGylation [30] or fusion to various protein scaffolds [31,32] to increase stability whilst maintaining the dimeric structure. The peptibody (AMG531/Romiplostim/Nplate, Amgen) now in clinical use was generated by fusion of four TPO mimetic peptides, via glycine bridges, to the Fc region of the IgG1 heavy-chain [31,33]. The 60 kDa peptibody has a circulatory half-life of 120–160 h [33] and a single subcutaneous dose in healthy volunteers produces an

Abbott Laboratories

increase in platelet count that peaks at days 12–16 [33,34]. Several studies have shown that Romiplostim (1–10 mg/ kg) increased platelet counts to 50  109 L 1 in patients with ITP with an overall response rate of approximately 80% of patients [31,35] and a sustained response following weekly subcutaneous injection in 49% of patients [35]. This response is associated with a reduction in concomitant ITP medications and bleeding incidence/ severity. These findings have been confirmed in an extension study (142 patients, mean treatment duration 69 weeks [36]) and a recent pooled analysis of 8 longer-term clinical studies (229 patients, median treatment duration 54 weeks [37]). These longer-term studies show that romiplostim has been well tolerated with some patients now receiving therapy for up to 162 weeks. The results of recent studies are summarized in an excellent review (Ref. [25]). No cases of anti-TPO neutralizing antibody have been detected from immunological assessment of 236 treated subjects [38]. Romiplostim was approved by the United States (U.S.) Food and Drug Administration (FDA) in August 2008 for chronic ITP in adults who have not responded to traditional treatments. It is also being trialed in chemotherapy-induced thrombocytopenia, Hepatitis-C related thrombocytopenia, and MDS although these studies are at early phase I/II stage [25]. This success story provides validation of the use of peptide HGF mimetics, whilst also highlighting a number of limitations to this approach; it is unlikely that peptide lead molecules will be developed as small molecule agonists with oral availability given the poor pharmacokinetic properties of peptides (i.e. rapid urinary excretion) and the necessity to increase potency and stability. Approaches to modifying peptide chemistry for reduced enzymatic cleavage in the circulation and to reduce clearance have been described [39] but it is likely that for optimal bioactivity HGF mimetic peptides will still need to be dimers and/or presented on a scaffold. Orally available, small molecule mimetics appear more likely to be identified using approaches that involve screening of large complex chemical libraries in cell based assays. This approach generated a number of TPO mimetic

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molecules [40,41] including the lead thrombopoietic small molecule, SB497115 (442 Da), that has been developed as eltrombopag [42] (marketed as Promacta1/Revolade1; GlaxoSmith Kline). This is a highly species-specific compound, which acts by binding to the transmembrane domain of TPO-R [43]. This emphasizes that there are alternative mechanisms of receptor activation consistent with activating mutations that have been reported in the TM domain of TPO-R [44] and the GM-CSF receptor betasubunit [45]. These mechanisms can be exploited by novel chemical compounds and are identified on the basis of activity rather than binding to recombinant receptor. Such screens where biological activity is the primary screen have the advantage that the receptor is in its native conformation, which for many cytokine receptors is a preformed, unliganded dimer [46–48], or a higher-order complex potentially involving interactions with other receptors [49–51] or interacting proteins (e.g. CBAP [52]). These structures provide the primary contact point for the cytokine and functional screening allows detection of novel agents that induce activity through interactions with these higher order complexes and/or via conformational changes required for activity. Extensive clinical studies have now been performed with eltrombopag. Daily doses over 10 days in healthy volunteers produce a peak platelet response on day 16 with an increase in platelet count 1.5-fold above baseline, compared with 6-fold for romiplostim [25]. Clinical studies with eltrombopag have been summarized recently [25]. Briefly, in a short placebo-controlled phase I/II study of 118 chronic ITP patients platelet counts above 50  109 L 1 were achieved in >80% of patients on 50 or 75 mg per day and upon cessation of drug treatment platelet counts returned to baseline [53]. These results were confirmed in a second placebo-controlled study [54]. Some patients (25%) on eltrombopag longer-term (25 weeks, 25–75 mg per day, EXTEND Trial) maintained platelet levels above 50  109 L 1. Between 70 and 85% of patients showed clinical benefit (platelet count above 50,000 mL, reduction of concomitant ITP therapy, reduced bleeding symptoms) with mild/moderate adverse events reported in 72% of patients [31,55–57]. The long-lasting effect has been confirmed in a large placebo-controlled phase III study (RAISE, 197 patients). Eltrombopag treatment for 6 months in previously treated patients with chronic ITP (platelet counts less than 30,000 mL) showed that long-term eltrombopag treatment (25–75 mg once daily) increased platelet count, decreased bleeding symptoms and resulted in a decrease in other ITP therapies [58]. The novel mechanism of action of eltrombopag may contribute to the finding that it appears equally effective in refractory and non-refractory patients [57]. Finally it has been shown recently that intermittent use of eltrombopag produced consistent results with similar platelet increases in each cycle [59]. Eltrombopag (Promacta/Revolade) has also been

approved in 2008 by the U.S. FDA for second-line treatment of ITP patients. A range of other thrombopoietic agents are being developed including those that bind the HBS (Fab 59, Peg-TPOmp), other TPO non-peptide mimetics (STS-T4) [60], AKR-501 [61]; LGD-4665 [62,63], and TPO agonist antibodies (MA01G4344 [64]) or minibodies (VB22B sc (Fv)2 [65]. These agents are still in various stages of clinical development and will not be discussed further here; their properties have been reviewed recently by Kuter [25].

4. Erythropoietin mimetics EPO mimetic peptides were successfully isolated by a team at Scripps, Affymax and Johnson Pharmaceutical Research Institute after screening phage display libraries for binders to the extracellular domain of EPOR. An initial peptide, EPO mimetic peptide 1 (EMP-1) [66], with no homology to EPO, was found to be bioactive as a disulphide-linked dimer, however, it was 10,000-fold less potent than rHuEPO in in vitro proliferation assays [46,67,68]. Further maturation of this initial peptide involving addition of flanking sequences and internal mutagenesis generated a 20-residue peptide with a Kd of 200 nM for EPOR and erythropoietic activity in vitro and in vivo [69]. Chemical modification through covalent linkage of polyethylene glycol generated the EMP1related di-peptide HematideTM (AffyMax) [70] which has increased half-life in the circulation and allows monthly administration. HematideTM has been shown to be safe in healthy volunteers with a one time intravenous injection (up to 0.1 mg/kg) increasing hemoglobin levels for 1 month [71]. Thus HematideTM shows promise as an alternative to the current forms of rHuEPO and may provide a response of longer duration than the recombinant HGF [71]. Phase I trials for CKD [71] indicate that HematideTM is safe and effective [72] and possibly active at lower doses than in healthy volunteers [71]. This new agent is currently undergoing phase III studies for CKD and phase II for cancer patients. The phase III trial for HematideTM will involve a large number of chronic renal failure patients in the U.S. and Europe. Trials in nondialysis patients, called PEARL 1 and PEARL 2, are also evaluating the safety and efficacy of HematideTM compared to Darbepoetin alfa. In dialysis patients, the trials, called EMERALD 1 and EMERALD 2, are evaluating HematideTM safety and efficacy and its ability to maintain corrected hemoglobin levels when patients are switched from epoetin alfa or epoetin beta. Some recent data [72] suggest that increased haemoglobin levels are sustained for at least 1 month, potentially providing a significant advantage over rHuEPO. Again the developmental path for this new clinical agent demonstrates the requirement for peptide dimerization and chemical

M. Perugini et al. / Cytokine & Growth Factor Reviews 20 (2009) 87–94

modification (final MW 60 kDa) and emphasizes that this approach is unlikely to be amenable to generation of small molecules that have oral availability. As with TPOR above, a number of other EPOR agonist molecules have been reported that do not appear to act through the HBS. A promising candidate for therapeutic development is the EPOR monoclonal antibody that is reported to act through a novel binding site, inducing activation by antibody-imposed conformational change [73]. This potent, fully human, agonistic antibody (ABT007) is produced in XenoMouse (XenoMouse XG2; Amgen) and has long serum half-life [74]. Given that antibodies have impressive track records as safe and highly effective therapeutics it is possible that generation of agonist antibodies will represent a useful approach to isolation of further HGF mimetics. Interestingly agonist antibodies have now been reported for TPOR (see above) and growth hormone receptor [75,76].

5. Development of further cytokine mimetics With the two mimetics for TPO now reaching the clinic there is likely to be new impetus for the mimetic approach. The field has advanced considerably with better functional screening approaches and high throughput technologies, isolation of agonist antibodies, and improved technologies for peptide presentation and half-life and hence there is potential now for generation of further cytokine mimetics. A strong candidate for this would be G-CSF, which is already in widespread use clinically. That small molecule G-CSF receptor agonists can be isolated has been demonstrated previously with isolation of the low potency synthetic compound, SB247464 [77] from an organic compound library. This compound bound the HBS of the mouse but not HuGCSFR and was not developed further. To date other G-CSF receptor agonist molecules or antibodies have not been described. The growth hormone (GH) receptor is a member of the cytokine receptor superfamily and also a candidate for generation of novel agonists. GH has been used as a therapy for individuals with idiopathioc short stature (ISS) for more than 20 years [78]. Since 2003 recombinant human growth hormone (rHuGH, Saizen) has been used to treat pediatric patients with short stature (predominantly ISS) (reviewed in Ref. [79]). This GH replacement therapy requires daily subcutaneous injections. Whilst a needle-free device has been developed to facilitate treatment [79] the treatment is associated with high cost. A number of approaches (e.g. PEGylation, sustained release formulation, ligand-receptor fusion proteins) have been utilized to improve therapy (reviewed by Birzniece et al. [80]), however, there is a need for novel therapies that minimize cost and provide for easy administration. GH therapy also has a potential role in other clinical

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settings, including wasting associated with prolonged critical illness [81] or HIV [82], HIV-associated lipodystrophy [83], inflammatory bowel disease [84] and juvenile idiopathic arthritis [85,86].

6. Conclusions The generation of HGF mimetics has proven a successful approach to generation of new thrombopoietic agents generating two new therapies that increase platelet count in healthy humans as well as in the majority of patients with ITP. The oral delivery of eltrombopag is a significant advantage and this represents a first class therapeutic agent that may lead the way for other small molecule cytokine mimetics. It is significant that both TPO mimetics have recently been approved by the U.S. FDA for use in ITP patients. More extensive use will further establish the efficacy and more clearly define any adverse effects associated with use of these new agents. Both agents have potential use in other clinical settings, including cancer therapy. The clinical assessment of the EPO mimetic HematideTM is not as far advanced but phase III trials will reveal whether it provides significant advantages over the current forms of rHuEPO with regard to reduced immunogenicity and reduced frequency of administration. These successes provide a paradigm for the development of HGF mimetics and advances in the field over the last decade suggest that HGF and growth hormone mimetics will continue to impact on disease treatment.

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[83] Benedini S, Terruzzi I, Lazzarin A, Luzi L. Recombinant human growth hormone: rationale for use in the treatment of HIV-associated lipodystrophy. Bio Drugs 2008;22(2):101–12. [84] Ahmed SF, Wong JS, McGrogan P. Improving growth in children with inflammatory bowel disease. Horm Res 2007;68(Suppl 5):117–21. [85] Simon D. Management of growth retardation in juvenile idiopathic arthritis. Horm Res 2007;68(Suppl 5):122–5. [86] Simon D, Prieur AM, Quartier P, Charles Ruiz J, Czernichow P. Early recombinant human growth hormone treatment in glucocorticoidtreated children with juvenile idiopathic arthritis: a 3-year randomized study. J Clin Endocrinol Metab 2007;92(7):2567–73. Dr. Michelle Perugini’s research experience relates to receptor signalling pathways, genetic mechnanisms that control normal myeloid cell growth and differentiation, and biochemical changes associated with myeloid leukaemia. Her doctor of philosophy degree (PhD), awarded in 2007 (The University of Adelaide, Department of Medicine), involved characterising the signalling properties of activated mutants of the GMCSF receptor. She is currently a post-doctoral research scientist in the Acute Leukaemia Laboratory at the Centre for Cancer Biology/Hanson Institute in Adelaide, Australia. Since completing her PhD, she has extended her focus to the signalling properties associated with activated FLT3 mutants in acute myeloid leukaemia. A recent first-author publication in Leukemia (in press) has identified the tumour suppressor Gadd45a as a commonly downregulated gene in response to FLT3 activated mutants, a pathway that contributes to the leukemic phenotype. Dr. Perugini has also been the recipient of The University of Adelaide post-graduate travelling fellowship (ISEH, 2005) and a Cancer Council of South Australia (TCCSA) travel grant in 2007 (ISEH). Dr. Antiopi Varelias was previously a Queen Elizabeth Hospital Research Foundation postdoctoral research fellow at the Women’s and Children’s Health Research Institute and The Queen Elizabeth Hospital in the Haematology and Oncology Department, where she focused on cytokine biology. She has since taken up a postdoctoral position with Dr. Geoff Hill at the Queensland Institute of Medical Research and is now studying transplant immunology.

Timothy Sadlon is a senior post-doctoral researcher at the Women’s and Children’s Health Research Institute and The University of Adelaide, Department of Paediatrics. He has extensive experience in molecular biology and interest in the regulation of haematopoietic stem cells and regulatory T cells.

Richard J. D’Andrea has been awarded the QEII Fellowship (Australian Research Council) (1989), the HM Lloyd Senior Research Fellowship in Oncology (1998) and the Peter Nelson Leukaemia Research Fellowship (2003). Until recently he was a program leader and deputy director at the Women’s and Children’s Health Research Institute (WCHRI). In 2005 he was appointed chief medical scientist in the Haematology and Oncology Division at the Queen Elizabeth Hospital (TQEH) and now runs a joint research group in the Centre for Cancer Biology (SA Pathology and Hanson Institute) and the TQEH with a focus on hematological diseases. He is currently an affiliate associate professor at the University of Adelaide, a full Member of the Hanson Institute, a Member of the Centre for Stem Cell Research (University of Adelaide) and a principal research fellow at WCHRI. He has a long-standing interest in cytokine receptors, having isolated and characterized a number of constitutively activated forms of the common beta chain (hbc) for IL-3, GM-CSF and IL-5. The concentration of activating lesions in the membrane-proximal extracellular domain showed that structural changes and interactions involving this domain were crucial for receptor activation, consistent with mutagenesis and structural studies, and led to a successful strategy for the isolation of cytokine receptor antagonists. Other research programs in his laboratory are focused on the molecular pathogenesis of myeloproliferative disease (MPD) and AML.