review

Antibody therapy for acute myeloid leukaemia Robin E. Gasiorowski,1,2 Georgina J. Clark,1 Kenneth Bradstock1,3 and Derek N. J. Hart1 1

ANZAC Research Institute, University of Sydney, 2Department of Haematology, Concord Cancer Centre, Concord Repatriation General Hospital, Concord, and 3Blood and Marrow Transplant Service, Westmead Hospital, Westmead, NSW, Australia

Summary Novel therapies with increased efficacy and decreased toxicity are desperately needed for the treatment of acute myeloid leukaemia (AML). The anti CD33 immunoconjugate, gemtuzumab ozogamicin (GO), was withdrawn with concerns over induction mortality and lack of efficacy. However a number of recent trials suggest that, particularly in AML with favourable cytogenetics, GO may improve overall survival. This data and the development of alternative novel monoclonal antibodies (mAb) have renewed interest in the area. Leukaemic stem cells (LSC) are identified as the subset of AML blasts that reproduces the leukaemic phenotype upon transplantation into immunosuppressed mice. AML relapse may be caused by chemoresistant LSC and this has refocused interest on identifying and targeting antigens specific for LSC. Several mAb have been developed that target LSC effectively in xenogeneic models but only a few have begun clinical evaluation. Antibody engineering may improve the activity of potential new therapeutics for AML. The encouraging results seen with bispecific T cell-engaging mAb-based molecules against CD19 in the treatment of B-cell acute lymphobalstic leukaemia, highlight the potential efficacy of engineered antibodies in the treatment of acute leukaemia. Potent engineered mAb, possibly targeting novel LSC antigens, offer hope for improving the current poor prognosis for AML. Keywords: acute myeloid leukaemia, antibody therapy, novel leukaemia drugs, radiotherapy. Understanding of the pathogenesis of acute myeloid leukaemia (AML) has advanced considerably with use of modern molecular techniques, such as whole genome sequencing (Ley, 2013), leading to improved risk stratification and the potential for truly personalized therapy. Whilst advances in the treatment of acute promyelocytic leukaemia (APL) have

Correspondence: Robin Gasiorowski, Dendritic Cell Biology and Therapeutics Group, ANZAC Research Institute, University of Sydney, Hospital Road, Concord, NSW 2139, Australia. E-mail: [email protected]

ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 164, 481–495

led to overall survival (OS) rates of over 90% (Iland et al, 2012), the current outcomes for most other patients with AML, particularly the elderly, are unsatisfactory. Although 75% of patients 0
5 9 109/l) and platelet recovery (>25 9 109/l) were 40 and 36 d respectively. The incidence of grade 3 or 4 sepsis and bleeding were significantly lower at 17% and 13% respectively. Whilst 29% of patients developed grade 3 or 4 hyperbilirubinaemia, this resolved with no medical intervention in most cases. Sixteen episodes of veno-occlusive disease occurred, although only two of these were in patients who had not undergone prior or subsequent HSCT. Unfortunately, the key phase 3 trial, South West Oncology Group Study S0106, failed to show a benefit when GO was added to induction chemotherapy for previously untreated AML (Petersdorf et al, 2013). In this study, 627 patients were treated with induction chemotherapy  6 g/m2 GO on day 4. There was no difference between the two arms in CR, CRp, OS or relapse-free survival (RFS). Preliminary data from this trial (Petersdorf et al, 2009) led to early trial closure and ultimately a commercial decision to withdraw the drug in 2010. Four other studies have been reported since then suggesting there may still be a role for GO in AML therapy. In the UK Medical Research Council (MRC) AML15 trial, 1113 patients with de novo or secondary AML were randomized to different intensive chemotherapy regimens (Burnett et al, 2011b). In addition they were randomized to 3 g/m2 GO on day one of induction, regardless of CD33 phenotype. For those patients who received GO in induction there was no overall difference in rate of relapse, OS or RFS. However a survival benefit for GO was seen in those with favourable risk cytogenetics (OS 79% vs. 51%) and a non-significant trend for benefit was noted for intermediate risk patients. Interestingly, CD33 expression level was not predictive of benefit. Fundamental investigations have shown that GO can affect CD33-negative cells (Bross et al, 2001; Schwemmlein et al, 2006) and GO was effective against CD33-negative ALL in a xenogeneic model due to GO uptake via CD33-independent endocytosis (Jedema et al, 2004). The MRC AML16 trial tested the addition of 3 mg/m2 GO on day 1 of induction chemotherapy with daunorubicin + cytarabine or daunorubicin + clofarabine in older patients with a median age of 67 years (Burnett et al, 2012). Patients who received GO had a statistically significant increase in RFS (21% vs. 16%, P = 0
04) and OS at 3 years (25% vs. 20%, P = 0
05) despite no differences in response rates. This benefit was not restricted to any particular subgroup, although the number of patients with favourable cytogenetics was low, reflecting the older age of the study cohort. 484

In the Acute Leukemia French Association (ALFA)-0701 trial, 280 patients aged 50–70 years with AML were randomized to receive GO 3 g/m2 on days 1, 4 and 7 plus induction chemotherapy with daunorubicin and cytarabine (Castaigne et al, 2012). At 2 years both event-free survival (EFS) (17
1% vs. 40
8%, P = 0
0003) and OS (53
2% vs. 41
9%, P = 0
0368) were improved in the GO arm. Subgroup analysis showed the benefit was restricted to those with a favourable or intermediate karyotype. Finally, in the Groupe Ouest Est d’Etude des Leucemies et Autres Maladies du Sang (GOELAMS) AML 2006 IR study, patients aged 18-60 years with AML and an intermediate risk karyotype were randomized to receive 6 mg/m2 GO with both induction and consolidation chemotherapy (Delaunay et al, 2011). The 3-year EFS was improved in the GO group of patients who could not receive allogeneic HSCT (53
7% vs. 27%, P = 0
0308), but there was no difference in OS. GO has been used in the treatment of APL, showing efficacy as a single agent in 14 of 16 patients with relapsed disease who had not been treated with arsenic trioxide (ATO) (Lo-Coco et al, 2004). In a study involving high risk APL [white blood cells (WBC) > 10
0 9 109/l], the 26 patients received GO in combination with all trans retinoic acid (ATRA) and ATO (Ravandi et al, 2009). With a median follow up overall of 99 weeks, the EFS for these patients was 65%, which compares to a 2-year EFS of 92% using ATO, ATRA and idarubicin in similar high risk patients (Iland et al, 2012). The Southwestern Oncology Group (SWOG) 0535 trial is evaluating the combination of ATRA, ATO and GO in patients with high risk APL. Taken together, these results suggest that GO may improve outcomes in patients with AML, particularly in those patients with favourable cytogenetics. The discrepancy between the recent positive GO trials and the S0106 trial needs to be explained. Firstly, the mortality in the S0106 control arm was unusually low at 0
8% compared to 5
8% in the GO arm (Petersdorf et al, 2013). Secondly, it is also notable that the four trials with favourable results used a lower or fractionated dose of GO resulting in overall less haematological and liver toxicity. Thirdly, patients who received GO in the S0106 trial received a lower dose of daunorubicin, 45 vs. 60 mg/m2 and this may have abrogated any beneficial effect of the GO. The recent more positive GO trial results have certainly rekindled interest in potential mAb therapies for AML.

Other CD33 antibodies Several groups have developed bispecific T-cell engager antibodies (BiTEs) targeting CD33. Aigner et al (2013) generated a CD33/CD3 antibody able to recruit T cells causing lysis of AML cells in vitro and showed effective inhibition of tumour growth in a xenogeneic model. Arndt et al (2013) reported on a fully humanized bispecific CD33/CD3 mAb, which was effective in both in vitro and in vivo models. ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 164, 481–495

Review Bispecific antibodies have been developed that target both CD16 and CD33 (Singer et al, 2010). These antibodies show enhanced in vitro ADCC against both AML cells lines and primary AML samples. Interestingly, a single-chain Fv triple body (sctb), consisting of two CD33 scFvs and one central CD16 scFv, was up to 200 times more potent at inducing lysis of AML cell lines than the bispecific format. However, this effect was not seen with primary AML samples. This group has generated further sctb constructs (Kugler et al, 2010) including a sctb targeting both CD123 and CD33 with a further binding site for CD16 on effector NK cells and macrophages. A CD33 scFv conjugated to a modified Pseudomonas toxin A (Schwemmlein et al, 2006) showed in vitro efficacy targeting AML cell lines and primary AML samples. A similar approach using moxetumomab pasudotox, a CD22 scFv conjugated to a truncated Pseudomonas toxin, has shown promising results in a phase 1 trial in patients with relapsed/ refractory hairy cell leukaemia (Kreitman et al, 2012) . A CD33 scFv conjugated to liposomes containing siRNA against the RUNX1-RUNX1T1 fusion protein has been reported (Rothdiener et al, 2010). These liposomes showed a modest inhibitory effect on AML cell lines in vitro. Another group have produced an anti-CD33 scFv conjugated to soluble tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) (ten Cate et al, 2009). This construct showed in vitro cytotoxicity against AML cell lines and primary AML cell lines. CD33 has also been targeted by transfecting Epstein-Barr Virus-specific cytotoxic T cells with an anti CD33 chimeric antigen receptor (CAR) (Dutour et al, 2012). These anti-CD33 CAR cells are able to kill CD33 + KG-1 cells in vitro and were effective in slowing the subcutaneous growth of AML10 cells in NOD/SCID mice. Finally, a recent report described a humanized CD33 antibody that has been conjugated to a synthetic DNA crosslinking pyrrolobenzodiazepine (Kung Sutherland et al, 2013). This agent, SGN-CD33A, shows impressive activity against multidrug-resistant cell lines, both in vivo and in vitro, that were unaffected by treatment with GO. A number of studies have suggested that treatment failures with GO may be due to P-glycoprotein (PGP)-mediated drug efflux from AML blasts (Naito et al, 2000; Walter et al, 2007), although in the AML15 trial there was no relationship between PGP expression and response to GO (Burnett et al, 2011b). A phase 1 trial of SGN-CD33A has recently opened (ClinicalTrials.gov Identifier: NCT01902329) and results are eagerly awaited.

Alternative antibody targets in AML Several groups have investigated alternative targets that bind mAb to AML blasts with greater specificity, i.e., to include LSC and avoid healthy myeloid cells. A summary of the clinical experience with GO, alternative CD33 mAb and the expanding number of mAb targeting novel antigens is shown in Table II. ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 164, 481–495

Other antigen targets tested in clinical studies CD45 CD45, or common leucocyte antigen, is a transmembrane protein expressed on all haematopoietic cells and stem cells, with the exception of erythrocytes. Its broad expression has limited CD45 as an antibody target in conventional treatment, however researchers in Seattle have used a variety of radiolabelled CD45 mAb in conditioning regimens for allogeneic HSCT (Orozco et al, 2012). In a phase 1/2 trial, 59 patients with AML in first CR (CR1) were treated with the CD45 antibody BC8 conjugated to iodine 131, busulfan and cyclophosphamide (BU/CY) as conditioning for allogeneic HSCT (Pagel et al, 2006). The 3-year disease-free survival was 61%, with a trend towards improved OS when compared to historical controls (hazard ratio 0
65), although this was not statistically significant (P = 0
09). Toxicities were similar to that seen with BU/CY alone, with mild infusion reactions and no evidence of delayed engraftment. However all patients developed at least grade 2 mucositis and hypothyroidism. In a separate phase 1 trial involving patients with AML or high risk myelodysplastic syndrome (MDS), 58 patients received 131 I-BC8, fludarabine and 2 Gy total body irradiation (TBI) as part of a reduced intensity conditioning regimen prior to allogeneic HSCT (Pagel et al, 2009). In this high-risk cohort, 86% of patients had >5% blasts at time of HSCT and the one-year OS was encouraging, at 40%. Again mild infusion reactions were noted, with no evidence of delayed engraftment. However, three patients did develop radiation-induced lung injury. A number of clinical trials are currently recruiting to examine this mAb, conjugated to either iodine 131 or yttrium 90, as part of conditioning regimens prior to allogeneic HSCT for AML, MDS and lymphoma (http://clinicaltri als.gov/ct2/results?term=bc8&recr=Open). Unfortunately, the technical difficulties in manufacturing radiolabelled mAbs mean that their use is likely to remain restricted to specialist centres.

CD66 CD66 is expressed on normal myeloid cells from the promyelocyte stage but is not generally expressed on AML blasts (Carrasco et al, 2000). Nevertheless, radiolabelled CD66 antibodies have been used to treat patients with AML as part of conditioning regimens prior to allogeneic HSCT. These agents rely on a ‘crossfire effect’, binding to bystander cells and emitting radiation to kill nearby leukaemic cells. MAb targeting CD66 are therefore only effective in patients with a substantial proportion of healthy myeloid cells and hence trials have been limited to patients with a least a partial remission. In a phase 1/2 study including 34 patients with high-risk AML (beyond CR1, adverse cytogenetics, or primary refractory disease) patients received rhenium 188 (188Re)-conjugated CD66 mAb prior to conditioning with 485

486 – Cell line

Cell line + Primary AML –

Cell line

– Primary AML + cell line

Primary AML + cell line Primary AML + cell line

Primary AML + cell line

Gemtuzumab

SGN-CD33A

Various

AMG330, bsAb CD33-3 131 I-BC8

Various

7G3

IMC-EB10

IPH2101 Bevacizumab

CD123

FLT3

KIR VEGF

Bi-HuM195

CD66

CD45

CD33/ CD123/ CD16 CD33/ CD3

213

N/A –

>80%

12/12 (100%)

2/29 (7%)

85–90%

N/A –

– –

Primary AML + cell line

Primary AML

–

–

12/12

–

–

Cell line + Primary AML – –

Primary AML

–

–

Cell line

90%

70–90%

Primary AML + cell line

Lintuzumab (HuM195)

CD33

AML

In vitro

Antibody

Target

Xenogeneic model

CD34+ CD38 fraction

Expression (%cases)

Table II. A summary of reported AML antibodies.

– –

Yes

–

–

–

–

–

–

–

Serial transplantation experiment

Efficacy

Phase I trial terminated due to lack of efficacy Safety shown in phase I trial Ineffective when combined with chemotherapy in previously untreated AML

CR in 1/40 patients in phase I trial

Promising results in phase I/II trials when used with conditioning therapy prior to allogeneic transplant Phase I/II trials have established safety and feasibility

–

–

Ineffective when combined with chemotherapy in relapsed/ refractory AML CR +CRp in 16% patients in phase I/II study Initial phase III trial showed excess toxicity and lack of efficacy. Four more recent studies suggest improved OS, at least in some subgroups. Some efficacy in relapsed APL. Role upfront remains unclear. –

Clinical trials

Bunjes et al (2001), Carrasco et al (2000), Ringhoffer et al (2005) Jin et al (2009), Roberts et al (2010) NCT00887926, Piloto et al (2005) Vey et al (2012) Ossenkoppele et al (2012)

Pagel et al (2006, 2009)

Aigner et al (2013), Arndt et al (2013)

Kung Sutherland et al (2013) Kugler et al (2010)

Burnett et al (2011b, 2012), Castaigne et al (2012), Delaunay et al (2011), Hamann et al (2002), Petersdorf et al (2013)

Rosenblat et al (2010)

Feldman et al (2005), Sutherland et al (2009)

References

Review

ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 164, 481–495

ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 164, 481–495

Campath-1H

H90

Various

Various

1075
7 ATIK2a IREM-1, MMRI-23

ESK1

CD52

CD44

CD47

CD96

CLL-1 TIM-3 CD300f

WT1

12/22 (54
5%) 28/34 13/24 (54
2%)

>75%

>75%

19/29 (66%)

–

45/52 (86
5%) 32/34 39/54 (72%)

13/13 (100%)

Primary AML + cell line

Primary AML + cell line Cell lines Primary AML + cell line Cell line Primary AML Primary AML + cell line ALL cell lines only

–

Primary AML

–

No apoptosis detected of primary AML Cell line

Primary AML

Primary AML + cell line

– 8/8 (100%)

Cell line

In vitro

13/13 (100%)

8/8 (100%)

45/124 (36%)

AML

Xenogeneic model

CD34+ CD38 fraction

Expression (%cases)

No

– Yes –

–

Yes – local engraftment only Yes

–

Serial transplantation experiment

Efficacy

–

– – –

Bergmann et al (1997), Dao et al (2013), Saito et al (2010)

Hosen et al (2007), Mohseni Nodehi et al (2012) Zhao et al (2010, 2012) Kikushige et al (2010) Korver et al (2009), Modra et al (2006)

Majeti et al (2009)

– –

Saito et al (2011), Tibes et al (2006) Jin et al (2006)

References

2/9 patients achieved CRp in one small study –

Clinical trials

AML, acute myeloid leukaemia; APL, acute promyelocytic leukaemia; CR, complete response; CRp, complete response with partial haematological recovery; OS, overall survival.

Antibody

Target

Table II. (Continued)

Review

487

Review 12 Gy TBI or CY/BU and HSCT (Bunjes et al, 2001). 18/34 (53%) of patients were alive in CR at a median follow up of 18 months. Whilst initial toxicities were not substantially greater than expected with conventional conditioning, five patients developed late renal toxicity, probably due to the cumulative radiation dose from TBI and the 188Re CD66 mAb. A subsequent phase 1/2 study by the same group used either 188Re-CD66 mAb or yttrium 90-conjugated CD66 mAb, again as part of conditioning prior to allogeneic HSCT in 20 patients aged 55–65 years with AML or MDS. Treatment was well tolerated with no radiation nephropathy, perhaps because TBI was not used for these patients. This cohort had a one-year OS of 70%, although by 30 months this had dropped to 17% (Ringhoffer et al, 2005).

CD123 CD123 is the alpha subunit of the interleukin 3 (IL3) receptor and binding of IL3 to this receptor leads to increased cellular survival and proliferation (Miyajima et al, 1993). Several studies have shown increased expression of CD123 by AML, including CD34 + CD38- LSCs, with lower levels of expression on normal HSCs, monocytes, endothelial cells and plasmacytoid dendritic cells (Jordan et al, 2000; Munoz et al, 2001; Riccioni et al, 2004; Florian et al, 2006; Jin et al, 2009). The experience to date with the CD123 mAb 7G3, is particularly informative as both detailed preclinical data from animal experiments and preliminary results from a phase 1 clinical trial with a chimeric variant of 7G3, CSL360, are available (Jin et al, 2009; Roberts et al, 2010). Incubation of AML samples with 7G3 reduced engraftment in NOD/SCID mice in 10 of 11 cases (Jin et al, 2009). Treatment with 7G3 reduced the ability of AML cells from primary engrafted mice to engraft secondary mice in serial transplantation experiments. This suggests that the mAb targets the LSC effectively. Importantly, 7G3 was much less effective when given to mice at day 28 post-transplantation, when the leukaemia burden was higher. In this instance, only two of five cases showed a bone marrow response. The chimeric CD123 mAb CSL360 was used to treat 40 patients with relapsed/refractory or high-risk AML in a phase 1 study (Roberts et al, 2010). Whilst in vitro studies showed effective targeting of CD123, the clinical effects were disappointing; only 1 patient achieved CR and no definite antileukaemic effect was seen in the other patients. The lack of efficacy in human subjects with a high tumour burden is perhaps unsurprising, given that treatment with 7G3 of NOD/SCID mice with established AML was much less effective than pre-incubation of AML samples with the mAb. This suggests a more successful therapeutic strategy may be to target patients with low tumour burden e.g. as part of consolidation therapy. Indeed this is the approach being taken in a phase 1 trial of CSL362, a new CD123 mAb engineered for increased ADCC (Clinical Trials.gov identifier: NCT01632852). 488

Others have constructed potential therapeutics based on single-chain Fv antibody fragments against CD123. A CD123 scFv generated from the 7G3 mAb has been used to manufacture an anti-CD123 chimeric antigen receptor that was subsequently transduced into cytokine induced killer (CIK) cells (Tettamanti et al, 2013). These transduced CIK cells showed in vitro activity against AML cell lines and blasts. Whilst these CIK cells also induced some lysis of CD123low monocytes and endothelial cells, this was no more than that seen with CD123-negative cells, suggesting that the off-target toxicity may be manageable. Stein et al (2010) developed an immunotoxin, a scFv fused to modified Pseudomonas Exotoxin A, and a bispecific scFv directed against CD123 and CD16. These agents showed promising in vitro efficacy on AML cell lines at nanomolar and picomolar concentrations, respectively, but clinical trials are awaited. Finally, CD123 has also been targeted using an IL3 fusion protein conjugated to diphtheria toxin. Whilst this agent showed some activity against TF-1 cells growing in SCID mice (Black et al, 2003), a phase 1 trial in 40 patients with AML, 35 of whom had relapsed /refractory disease, was disappointing with only one CR observed (Frankel et al, 2008).

FLT3 The FMS-like tyrosine kinase 3 receptor (FLT3) plays an important role in the survival, differentiation and proliferation of haematopoietic cells. Internal tandem duplication mutations of FLT3 occur in around 30% of patients with AML, result in constitutive activation of FLT3, and are associated with a poor prognosis (Leung et al, 2013). Whilst a number of small molecule functional tyrosine kinase (TK) inhibitors have been developed, their activity as single agents targeting FLT3 appears limited to reduction of the peripheral blood blast count (Swords et al, 2012). FLT3 is expressed on leukaemic blasts in approximately 80% of cases of AML (Rosnet et al, 1996) making it a suitable target for mAb. The fully humanized mAb IMC-EB10 is able to block FLT3 activation in vitro and showed promising activity in NOD/SCID mice engrafted with an AML cell line and primary AML blasts (Piloto et al, 2005). However, a phase 1 trial of this agent was terminated in 2012 due to lack of efficacy (ClinicalTrials.gov Identifier: NCT00887926). Hofmann et al (2012) used glyco-engineering to optimize the Fc portion of the FLT3 mAb, 4G8SDIEM, with enhanced in vitro ADCC. The authors mentioned a transient clearance of blasts from the peripheral blood of a patient treated with this mAb but no further information on its clinical efficacy has been published.

KIR mAb Killer inhibitory receptors (KIRs) are expressed on the surface of NK cells and prevent activation of NK cells when they bind to their ligands, major histocompatibility complex ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 164, 481–495

Review (MHC) class I alleles (Farag et al, 2002). NK cells pre-treated with the anti-KIR mAb, IPH2101 (formerly known as 17F9), showed superior in vitro cytotoxicity to autologous AML blasts (Romagne et al, 2009). Treatment of NOD/SCID mice with this same anti-KIR mAb, prior to infusion of AML blasts and human leucocyte antigen (HLA)-C matched NK cells, resulted in superior survival compared to untreated mice. A phase 1 trial of an anti-KIR mAb, IPH2101, in elderly AML patients in first CR showed in vitro evidence of effective NK cell activation with this agent (Vey et al, 2012). Treatment was well tolerated with no haematological toxicity and some, generally mild, infusion reactions. Patients who were treated with the higher doses of the mAb, with subsequent saturation of KIRs, had significantly better OS compared to those treated with the lower dose (P = 0
03).

suggested as a cancer stem cell marker in both haematological and epithelial malignancies. One of the first attempts to target the LSC was with H90, an antibody directed against the cell adhesion molecule CD44 (Jin et al, 2006). This antibody was able to inhibit engraftment of human AML cells in NOD/SCID mice, when given from day 10 post-transplantation but was not effective in mice with established AML. Human AML cells taken from H90-treated NOD/SCID mice were unable to engraft secondary mice, suggesting successful eradication of the LSC. Bivatuzumab mertansine, an immunoconjugate targeting CD44v6, used to treat squamous cell carcinoma of the head, neck and oesophagus, was withdrawn after a patient developed fatal skin toxicity, probably due to expression of CD44v6 in skin keratinocytes, in a phase 1 trial (Tijink et al, 2006). Concerns over potential toxicities caused by its broad expression may limit the use of CD44 as a target in AML.

VEGF (Bevacizumab)

CD47

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis in healthy cells. It has been shown to promote leukaemic cell survival (Ding et al, 2012a) and patients with AML have increased numbers of blood vessels compared to healthy controls (de Bont et al, 2001). Bevacizumab is a humanized mAb, which neutralizes all human VEGF-A isoforms (Ferrara et al, 2004). However, in a randomized phase 2 study of 171 patients aged >60 years with newly diagnosed AML, the addition of bevacizumab to induction and consolidation chemotherapy failed to improve CR, EFS or OS (Ossenkoppele et al, 2012).

CD47 is a ubiquitously expressed transmembrane protein suggested to act as a ‘don’t eat me’ signal. It acts as the ligand for signal regulatory protein a (SIRPa) on cells such as dendritic cells and macrophages to inhibit phagocytosis. MAb have been developed to block this signal, allowing macrophages to engulf tumour cells, although some groups have expressed doubt that this mechanism fully explains the effect of CD47 antibodies (Soto-Pantoja et al, 2012; Zhao et al, 2012). Nonetheless, there is higher expression of CD47 in the LSC compartment than in normal HSC and increased expression of CD47 is a poor prognostic factor in patients with AML (Majeti et al, 2009). CD47 antibodies enable phagocytosis of LSC by macrophages but do not directly cause apoptosis. A CD47 blocking mAb was effective in mice with established AML engraftment and was able to prevent subsequent engraftment in serial transplantation experiments. CD47 is upregulated in a number of malignancies and CD47 antibodies are effective in xenogeneic models of ALL (Chao et al, 2011), myeloma (Kim et al, 2012), non-Hodgkin lymphoma (Chao et al, 2010), leiomyosarcoma (Edris et al, 2012) and a variety of other solid tumours (Willingham et al, 2012). However, as it is expressed on healthy cells there is concern that therapies directed at this target may cause unwanted off-target effects. A rat anti-mouse CD47 did not affect normal HSC or cause any significant metabolic derangement in mice but did result in a marked neutropenia (Majeti et al, 2009). CD47 has been shown to be downregulated on HSC from patients with haemophagocytic lymphohistiocytosis, enabling them to be engulfed by macrophages (Kuriyama et al, 2012). Further studies will help to address these safety concerns.

CD52 CD52 is a glycoprotein of unknown function expressed on the surface of mature lymphocytes, NK cells, monocytes, macrophages and some dendritic cells (DC). Alemtuzumab is an anti-CD52 mAb, used mainly in the treatment of haematological malignancies, such as chronic lymphocytic leukaemia, and for T cell depletion in HSCT. Saito et al (2011) showed some efficacy both in vitro and in a xenogeneic model. In a small case series of 9 patients with CD52-positive relapsed/refractory AML, two patients achieved CRp (Tibes et al, 2006). The authors examined CD52 expression in a larger cohort of AML patients and found expression in 45 of 124 cases (36%). This relatively low expression and the immunosuppressive side effects of the antibody are likely to limit the use of this agent in AML.

Antigen targets tested in preclinical models CD44 CD44 is a transmembrane glycoprotein involved in cell adhesion, survival, migration, and differentiation. It is expressed ubiquitously, with numerous splice variants, but has been ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 164, 481–495

CD96 CD96 is an immunoglobulin superfamily member that is expressed on activated T cells and NK cells as well as AML 489

Review blasts. It was expressed in the majority of CD34 + CD38leukaemia stem cells in 19/29 cases of AML and is expressed at much lower levels on normal HSC (Hosen et al, 2007). In addition, in 4 of 5 cases the CD96+ fraction from AML samples was able to engraft immunodeficient mice, whilst the CD96-fraction could not. Mohseni Nodehi et al (2012) demnostrated that anti-CD96 ‘mini-antibodies’, consisting of an affinity matured scFv fused to an IgG1 Fc region with enhanced CD16 binding, show greater in vitro ADCC of AML cell lines than an unmanipulated antibody.

CLL-1 C-type lectin-like molecule-1 (CLL-1) is an immunoregulatory transmembrane glycoprotein found on both healthy myeloid cells and approximately 85% of cases of AML (Zhao et al, 2010). It is present on CD34+ CD38 AML stem cells in around 50% of cases and is expressed by normal haematopoietic stem cells in a significant minority of cases, around 20%. A CLL-1 mAb was shown to have both in vitro against efficacy primary AML samples and modest in vivo efficacy in a mouse xenogeneic model (Zhao et al, 2010).

TIM-3 T cell immunoglobulin mucin-3 (TIM-3) is expressed on CD4 T cells and acts as a negative regulator of T-helper cell type 1 (Th1) responses. It is also found on DC, monocytes and macrophages. Kikushige et al (2010) showed that the TIM-3 fraction from AML patients is unable to engraft immunodeficient mice. Another group suggested that TIM-3 may be able to distinguish LSCs from normal HSCs (Jan et al, 2011). AML cells from mice treated with the TIM-3 mAb ATIK2a were unable to engraft secondary mice, suggesting effective targeting of the LSC.

CD300f The CD300a-f molecules are expressed on leucocyte membranes where, via paired triggering and inhibitory functions, they are able to modulate immune responses (Clark et al, 2009). CD300f, also known as immune receptor expressed on myeloid cells 1 (IREM-1), is an inhibitory receptor whose expression is restricted to myeloid cells (Alvarez-Errico et al, 2004). Crosslinking CD300f on macrophages has been shown to induce cell death via a caspase-independent mechanism (Can et al, 2008). These properties have led our group and others to investigate CD300f as a potential therapeutic target in AML (Modra et al, 2006; Korver et al, 2009). CD300f is expressed by AML blasts in approximately 75% of AML cases and on CD34 + CD38- LSC in around 50% (Korver et al, 2009). The chimeric CD300f mAb, D12, has shown in vitro efficacy against AML cell lines and primary AML samples and modest in vivo efficacy in NOD/SCID mice inoculated with both HL-60 cells and primary AML 490

cells. CD300f is internalized after binding with mAb making it logical to conjugate potential mAb to an anti-leukaemic toxin.

WT1 Wilms tumour 1 (WT1) is an oncoprotein that is upregulated in a wide variety of malignancies, including ovarian cancer, mesothelioma and in over 75% of cases of AML (Bergmann et al, 1997). As WT1 is intracellular, a number of groups have attempted to target WT1 with vaccination therapy rather than a mAb. This approach has yielded some encouraging results, most notably in a phase 1/2 trial of 10 patients with AML and evidence of molecular residual disease (Van Tendeloo et al, 2010). Vaccination with autologous DC loaded with WT1 mRNA resulted in molecular remission in 50% of the treated patients. Intracellular antigens are processed and peptide fragments presented by MHC molecules on the cell surface, where they are accessible to mAb. Dao et al (2013) recently reported a fully human IgG1 specific for the WT-1 peptide RMF/ HLA-A0201 complex. This mAb showed in vitro ADCC against AML cell lines and primary HLA-A2 + blasts. In vivo efficacy was demonstrated against ALL cell lines. This innovative approach using a mAb to target intracellular antigen has the potential to be effective in several malignancies, but each mAb will be necessarily confined to patients with a specific HLA subtype.

Conclusions and future prospects Novel effective therapies, with lesser toxicity, suitable for use in older patients, are desperately needed for the treatment of AML. There is growing evidence to suggest that the high relapse rates seen in AML are due to chemoresistant LSC. New mAb therapies targeting these cells would be an invaluable addition to therapeutic AML protocols. A number of recent studies suggest that GO may improve OS in AML, particularly in those patients with a favourable karyotype, although overall the reported improvements are relatively modest. Novel mAb have been developed, which appear to target LSC and can prevent secondary engraftment of AML cells in pre-clinical transplantation experiments but, to date, there is limited clinical evidence of the efficacy of these antibodies in humans. The anti-CD123 mAb, 7G3, reduces engraftment in serial transplantation experiments but has only limited efficacy in relapsed/refractory patients (Roberts et al, 2010). These differences may be partly due to the higher leukaemic burden in patients with relapsed/refractory disease but may also reflect limitations of the current assays used to define LSC. Given the large number of novel agents in preclinical testing, improved animal models will be an invaluable aid to progress. Most studies to date have used xenogeneic models generated from a very limited number of patient AML samples. Testing novel agents across a broader panel of AML samples would be advantageous. Current ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2014, 164, 481–495

Review models rely on the use of heavily immunosuppressed mice to allow engraftment and the mechanism of action of a mAb in this immune setting will differ from that in a healthy individual and a patient with AML. Mice may be engrafted with human CD34 + cells to generate a fully human haemopoietic system, prior to subsequent engraftment with AML. This approach may give a more useful approximation of the ultimate clinical effect of novel mAb, including their efficacy and probable haematological toxicity. The efficacy of novel mAb as single agents may be limited. In a disease with the rapid clinical tempo of AML, eliminating a proportion of the blasts is insufficient and will inevitably produce only transient clinical responses. Recent studies using whole genome sequencing suggest that several AML clones may be present at diagnosis and that different subclones emerge during disease progression (Ding et al, 2012b; Walter et al, 2012). Therefore combining novel mAb with traditional induction chemotherapy may be a more effective strategy, preventing the emergence of chemoresistant clones. Such combination mAb immuno-chemotherapeutic regimens will need to take into account possible overlapping toxicities. Unfortunately, CD33 and many of the novel antigens, are expressed on healthy myeloid cells so treatments targeting these will cause neutropenia. This toxicity may limit the use of an anti-AML mAb for prolonged maintenance therapy. In B-ALL the elimination of healthy B cells by drugs targeting CD19 or CD20 can be ameliorated by infusion of intravenous immunoglobulin but there is no such solution for a patient rendered profoundly neutropenic. To reduce overlapping toxicities, mAb may need to be used sequentially with traditional chemotherapy, perhaps immediately after the completion of induction and consolidation chemotherapy. NK cells from patients with AML have been shown to have lower levels of natural cytotoxicity receptors (NCRs) and lower levels of in vitro cytolytic activity against autologous leukaemia cells (Costello et al, 2002). However patients who achieve CR upregulate these NCRs resulting in restored cytotoxicity (Fauriat et al, 2007). Therefore it is possible that mAb, which rely on ADCC, may be more effective when used after induction therapy. Interestingly, attempts to use GO, which does not rely on ADCC, as consolidation therapy have been unsuccessful. A study in

References Aigner, M., Feulner, J., Schaffer, S., Kischel, R., Kufer, P., Schneider, K., Henn, A., Rattel, B., Friedrich, M., Baeuerle, P.A., Mackensen, A. & Krause, S.W. (2013) T lymphocytes can be effectively recruited for ex vivo and in vivo lysis of AML blasts by a novel CD33/CD3-bispecific BiTE((R)) antibody construct. Leukemia, 27, 1107–1115. Alvarez-Errico, D., Aguilar, H., Kitzig, F., Brckalo, T., Sayos, J. & Lopez-Botet, M. (2004) IREM-1 is a novel inhibitory receptor expressed by

older patients, who had received intensive induction chemotherapy, showed no impact on relapse rates or OS (Lowenberg et al, 2010), whilst a second study in younger AML patients randomized to GO after high dose cytarabine consolidation and prior to autologous HSCT also showed no effect on OS (Fernandez et al, 2011). Whilst GO has been shown to be effective in ATO-naive APL patients with molecular relapse (Lo-Coco et al, 2004), its role in patients with molecular relapse after upfront ATO and ATRA is currently unclear. Effective treatment of AML with mAb will require a personalized, tailored approach. The incorporation of clinically validated antibodies into routine flow cytometry panels will allow rapid immunophenotyping of blasts and CD34+ CD38 ‘LSCs’. In addition, the inclusion of these novel markers may allow better characterization of the leukaemia immunophenotype and hence improved flow cytometric monitoring of minimal residual disease. These data, conventional cytogenetics and screening for molecular mutations will improve risk stratification and allow clinicians to make rational decisions regarding the best combination of treatments. Antibodies to novel AML antigens, engineered for maximum anti-leukaemic potency, will form a valuable part of the future therapeutic armoury.

Acknowledgements This work was supported by grants from the National Health and Medical Research Council and the Cancer Institute of NSW. Gasiorowski has received an Enid Ng Fellowship in Haematology. Gasiorowski and Hart conceived the project, wrote and reviewed the manuscript. Clark and Bradstock helped define the scope of the review, edited and approved the final manuscript.

Conflict of interest Hart and Clark hold intellectual property in relation to CD300f on behalf of the Dendritic Cell Biology and Therapeutics Group. Other intellectual property from the group is owned by the ANZAC Research Institute / University of Sydney.

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