COMMENTARY Molecular & Cellular Oncology 1:4, e969129; October/November/December 2014; Published with license by Taylor & Francis Group, LLC

Unexpected mechanisms of action for a receptor-blocking antibody

Timothy R Hercus1, Sophie E Broughton2, Matthew P Hardy3, Tracy L Nero2, Nicholas J Wilson3,*, Michael W Parker2,4,*, and Angel F Lopez1,* 1The Centre for Cancer Biology; SA Pathology and the University of South Australia; Adelaide, South Australia; 2Australian Cancer Research Foundation Rational Drug Discovery Centre; St. Vincent’s Institute of Medical Research; Fitzroy, Victoria; 3CSL Limited; Parkville, Victoria; 4Department of Biochemistry and Molecular Biology; Bio21 Molecular Science and Biotechnology Institute; University of Melbourne; Parkville, Victoria, Australia

SL362 is a humanized -3 receptor-transduced T cells (CART), IL-3: C(IL-3)-neutralizing monoclonal diphtheria toxin conjugates, and the thera- antibody with enhanced effector function peutic monoclonal antibody CSL362.6,7 a Keywords: antibodies, cancer, cytokine that binds the subunit of the IL-3 CSL362 is derived from the anti-IL-3 a receptor, signaling receptor (IL3R ). The crystal structure receptor blocking monoclonal antibody of an IL3Ra:CSL362 complex shows 7G3,8 which was humanized, affinity Abbreviations: ADCC, antibody-depen- that IL3Ra adopts “open” and “closed” optimized, and engineered via modifica- dent cell-mediated cytotoxicity; AML, conformations. CSL362 blocks IL-3 tions in the Fc-domain to have a higher acute myeloid leukemia; ALL, B-cell acute function through both IL3Ra conforma- affinity for CD16, resulting in enhanced lymphoblastic leukemia; CART, chimeric tions but via distinct and unexpected antibody-dependent cell-mediated cyto- antigen receptor-transduced T cells; mechanisms. toxicity (ADCC) activity (Fig. 1b and a CML, chronic myeloid leukemia; GMR , The cytokine interleukin-3 (IL-3) regu- 1c).7 CSL362 has been shown to deplete granulocyte-macrophage colony-stimulat- lates survival, proliferation, differentiation, both CML and AML blasts by autologous a ing factor receptor subunit; IL-3, inter- and activation of cells of the hemopoietic natural killer (NK) cells in vitro.7,9 More- a a leukin-3; IL3R , IL-3 receptor subunit; and immune system (Fig. 1a). IL-3 signals over, in mouse models of human AML a a IL5R , IL-5 receptor subunit; through a receptor comprising the IL-3 and CML, CSL362 reduced engraftment a a IL13R 1, interleukin-13 receptor sub- receptor a subunit (IL3Ra or CD123) of both human AML and CML stem a unit 1; IL13R 2, interleukin-13 receptor and the b common (bc) subunit.1,2 cells.7,9 The inhibitory effects on IL-3 a subunit 2; LSPC, leukemic stem and IL3Ra is overexpressed in a number of contributed to reduced AML engraftment progenitor cell; MDS, myelodysplastic hematological malignancies including and reduced AML LSPC survival, and syndromes; NK, natural killer cell; NTD, acute and chronic myeloid leukemia prevented IL-3–mediated rescue of dasati- N-terminal domain; TKI, tyrosine kinase (AML and CML respectively), myelodys- nib- or nilotinib-induced CML LSPC inhibitor. plastic syndromes (MDS), B-cell acute death. Importantly, however, CSL362 © Timothy R Hercus, Sophie E Broughton, Matthew lymphoblastic leukemia (ALL), hairy cell directly mediated killing of leukemic cells P Hardy, Tracy L Nero, Nicholas J Wilson, Michael W leukemia, and Hodgkin’s lymphoma. via NK cells.7,9 These studies have Parker, and Angel F Lopez Compared to normal hemopoietic stem prompted a phase I clinical trial of *Correspondence to: Nicholas J Wilson; Email; nick. a [email protected]; Michael W Parker; Email: cells, IL3R overexpression is also CSL362 in patients with AML (Clinical- [email protected]; Angel F Lopez; Email: angel. observed in leukemic stem and progenitor Trial.gov identifier: NCT01632852). [email protected] cells (LSPCs) from AML, CML, and We recently solved the crystal structure Submitted: 08/14/2014 MDS that represent a minor population of a binary complex comprising a CSL362 Revised: 08/20/2014 of cells that give rise to the bulk tumor, Fab fragment bound to a soluble form of but are also considered therapy-resistant IL3Ra (IL3Ra:CSL362).10 This study Accepted: 08/22/2014 and responsible for disease relapse.3,4 At revealed that the N-terminal domain http://dx.doi.org/10.4161/23288604.2014.969129 least in the case of AML, IL3Ra expres- (NTD) of IL3Ra could adopt distinct This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non- sion is inversely correlated with overall conformations with respect to domains 2 5 a Commercial License (http://creativecommons.org/ survival. Thus, IL3R represents an and 3 (D2 and D3) of the receptor, with 2 licenses/by-nc/3.0/), which permits unrestricted attractive therapeutic target for a number non-identical forms of the IL3Ra: non-commercial use, distribution, and reproduction of hematological malignancies for which CSL362 complex observed within the in any medium, provided the original work is prop- a range of agents are currently being crystal. The NTD of IL3Ra was posi- erly cited. The moral rights of the named author(s) have been asserted. developed, including chimeric antigen tioned in either a “closed” conformation www.landesbioscience.com Molecular & Cellular Oncology e969129-1 Figure 1. Dual mechanism of action of CSL362-induced cell death. The interleukin-3 (IL-3) receptor a subunit (IL3Ra) is overexpressed on various leuke- mic cells, including leukemic stem and progenitor cells (LSPCs), and is associated with poor prognosis in acute myeloid leukemia (AML). (a) IL-3-mediated receptor signaling results in formation of a complex between IL3Ra and the b common (bc) receptor (modeled on the granulocyte-macrophage colony- stimulating factor receptor structure2) that induces cell survival, differentiation, or proliferation. (b) CSL362 is a humanized anti-IL3Ra neutralizing mono- clonal antibody that has enhanced effector function and is currently in a phase I clinical trial in patients with AML. Neutralization of IL-3 signaling in leu- kemic cells enhances their susceptibility to tyrosine kinase inhibitor (TKI)-induced cell death and reduces the survival of LSPCs. CSL362 neutralizes IL-3- induced signaling by preventing IL-3 binding to IL3Ra (left) and by preventing higher order signaling complex formation (right). (c) In addition to its neu- tralizing activity, CSL362 also has enhanced effector function that results in potent natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC) killing of target cells, including AML blasts, chronic myeloid leukemia (CML) blasts, and LSPCs. or a novel “open” conformation. The of ligand binding and subsequent down- that were proposed to force IL3Ra to “closed” conformation closely resembled stream signaling. IL5Ra, IL13Ra1, adopt an “open-like” conformation, we the receptor conformation adopted by IL13Ra2, and the granulocyte-macro- showed that CSL362 and IL-3 can simul- related subunits, includ- phage colony-stimulating factor receptor taneouslybindtoanIL-3receptorcom- ing the interleukin-5 receptor a subunit a subunit, GMRa, also have a flexible plex where IL3Ra exists in an open-like IL5Ra and the interleukin-13 receptor a linker between the NTD and D2, suggest- conformation, but that the binding of subunits IL13Ra1 and IL13Ra2, when ing a similar mechanism of receptor CSL362 still blocks IL-3 receptor func- bound to ligand. The main difference modulation. tion.10 On the basis of these results we between the closed and open forms of the Our crystal structure revealed that proposed that IL-3 signaling arises through receptors was the angle between the NTD CSL362 binds exclusively to the IL3Ra the initial assembly of a 2:2:2, IL-3: and D2, which showed a difference of NTD through interactions involving the IL3Ra:bc hexamer complex that provides approximately 40. The open and closed BC, DE, and FG loops and strand D of high-affinity IL-3 binding. Subsequent forms of IL3Ra observed in the crystal the NTD.8,10 Investigation of the func- formation of a higher-order complex structure likely represent the dynamic tional IL-3 binding site in IL3Ra through that requires interactions between hex- extremes of potential NTD conforma- homology modeling and mutagenesis stud- amers allows bc-associated JAK2 activa- tions. A disulfide bond between C76 in ies indicated that the binding interactions tion and is essential for signal the NTD and C194 in D2 and a hydro- of CSL362 and IL-3 with IL3Ra are transduction. Such higher-order complex gen bond between S74 in the NTD and largely non-overlapping, and prompted formation was first reported for the GM- D196 in D2 place constraints on the further analysis to understand how CSF receptor and is potentially a com- degree to which the NTD can open, CSL362 is able to function as an antago- mon feature of the bc family of recep- whereas interactions between A72 in the nist of IL-3 function. In the closed IL3Ra tors.2 Importantly, CSL362 can inhibit NTD and F198 in D2 provide a complex, binding of CSL362 prevents IL- IL-3-induced receptor activation in “doorstop effect” to limit further closing 3 binding through steric interference either the classic closed form by directly of the IL3Ra. Our data demonstrated whereas in the open IL3Ra complex blocking IL-3 binding, or in the novel that a likely consequence of the flexibility CSL362 and IL-3 can potentially bind open form by preventing higher order in the NTD is to allow subtle modulation simultaneously. Using IL3Ra mutations complex formation (Fig. 1b).

e969129-2 Molecular & Cellular Oncology Volume 1 Issue 4 The dual mechanisms of CSL362- Operational Infrastructure Support myelogenous leukemia is associated with enhanced blast mediated IL-3 neutralization demonstrate Scheme to St Vincent’s Institute is proliferation, increased cellularity, and poor prognosis. Blood 2002; 100: 2980-8; PMID:12351411; http://dx. quite distinct structural explanations that acknowledged. MWP is an NHMRC doi.org/10.1182/blood-2002-03-0852 may be germane in understanding the Research Fellow and SEB is a Postdoctoral 6. Tettamanti S, Marin V, Pizzitola I, Magnani CF, Giordano Attianese GM, Cribioli E, Maltese F, Galim- blocking activity of other antibodies that Fellow supported by the Leukaemia Foun- berti S, Lopez AF, Biondi A, et al. Targeting of acute block function. dation of Australia. myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric anti- gen receptor. Br J Haematol 2013; 161: 389-401; PMID:23432359; http://dx.doi.org/10.1111/bjh. Disclosure of Potential Conflicts of Interest References 12282 1. Lopez AF, Hercus TR, Ekert P, Littler DR, Guthridge 7. Busfield SJ, Biondo M, Wong M, Ramshaw HS, Lee MWP and AFL are consultants for M, Thomas D, Ramshaw HS, Stomski F, Perugini M, EM, Ghosh S, Braley H, Panousis C, Roberts AW, He CSL Limited which is developing D’Andrea R, et al. Molecular basis of cytokine receptor SZ, et al. Targeting of acute myeloid leukemia in vitro CSL362 and have received research sup- activation. IUBMB Life 2010; 62: 509-18; and in vivo with an anti-cd123 mab engineered for PMID:20540154; http://dx.doi.org/10.1002/iub.350 optimal ADCC. Leukemia 2014; 28: 2213-21; port from CSL Limited. MPH and NJW 2. Broughton SE, Dhagat U, Hercus TR, Nero TL, Grim- PMID:24705479; http://dx.doi.org/10.1038/leu. are employees of CSL Limited. baldeston MA, Bonder CS, Lopez AF, Parker MW. 2014.128 The GM-CSFIL-3IL-5 cytokine receptor family: from 8. Sun Q, Woodcock JM, Rapoport A, Stomski FC, ligand recognition to initiation of signaling. Immunol Korpelainen EI, Bagley CJ, Goodall GJ, Smith WB, Rev 2012; 250: 277-302; PMID:23046136; http://dx. Gamble JR, Vadas MA, et al. Monoclonal antibody Acknowledgments doi.org/10.1111/j.1600-065X.2012.01164.x 7G3 recognizes the N-terminal domain of the human This research was partly undertaken at 3. Jordan CT, Upchurch D, Szilvassy SJ, Guzman ML, interleukin-3 (IL-3) receptor a-chain and functions as a Howard DS, Pettigrew AL, Meyerrose T, Rossi R, specific IL-3 receptor antagonist. Blood 1996; 87: 83- the Australian Synchrotron, Victoria, and Grimes B, Rizzieri DA, et al. The interleukin-3 recep- 92; PMID:8547680 we acknowledge the technical assistance of tor alpha chain is a unique marker for human acute 9. Nievergall E, Ramshaw HS, Yong AS, Biondo M, myelogenous leukemia stem cells. Leukemia 2000; 14: Busfield SJ, Vairo G, Lopez AF, Hughes TP, White the CSL Research Department. 1777-84; PMID:11021753; http://dx.doi.org/10.1038/ DL, Hiwase DK. Monoclonal antibody targeting of IL- sj.leu.2401903 3 receptor alpha with CSL362 effectively depletes CML Funding 4. Jin L, Lee EM, Ramshaw HS, Busfield SJ, Peoppl AG, progenitor and stem cells. Blood 2014; 123: 1218-28; Wilkinson L, Guthridge MA, Thomas D, Barry EF, PMID:24363400; http://dx.doi.org/10.1182/blood- This work was supported by grants Boyd A, et al. Monoclonal antibody-mediated targeting 2012-12-475194 from the National Health and Medical of CD123, IL-3 receptor alpha chain, eliminates human 10. Broughton SE, Hercus TR, Hardy MP, McClure BJ, acute myeloid leukemic stem cells. Cell Stem Cell 2009; Nero TL, Dottore M, Huynh H, Braley H, Barry EF, Research Council (NHMRC) of Australia, 5: 31-42; PMID:19570512; http://dx.doi.org/10.1016/ Kan WL, et al. Dual mechanism of interleukin-3 recep- from Cancer Australia and from the Aus- j.stem.2009.04.018 tor blockade by an anti-cancer antibody. Cell Rep 5. Testa U, Riccioni R, Militi S, Coccia E, Stellacci E, 2014; 8: 410-9; PMID:25043189; http://dx.doi.org/ tralian Cancer Research Foundation. Samoggia P, Latagliata R, Mariani G, Rossini A, Battis- 10.1016/j.celrep.2014.06.038 Funding from the Victorian Government tini A, et al. Elevated expression of IL-3Ralpha in acute

www.landesbioscience.com Molecular & Cellular Oncology e969129-3

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Hercus, TR; Broughton, SE; Hardy, MP; Nero, TL; Wilson, NJ; Parker, MW; Lopez, AF

Title: Unexpected mechanisms of action for a cytokine receptor-blocking antibody.

Date: 2014-10

Citation: Hercus, T. R., Broughton, S. E., Hardy, M. P., Nero, T. L., Wilson, N. J., Parker, M. W. & Lopez, A. F. (2014). Unexpected mechanisms of action for a cytokine receptor-blocking antibody.. Mol Cell Oncol, 1 (4), pp.e969129-. https://doi.org/10.4161/23288604.2014.969129.

Persistent Link: http://hdl.handle.net/11343/208962

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