Leukemia (2012) 26, 199–213 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu REVIEW

Monoclonal antibody-based therapy as a new treatment strategy in multiple myeloma

NWCJ van de Donk1, S Kamps1, T Mutis2 and HM Lokhorst1

1Department of Hematology, University Medical Center Utrecht, Utrecht, The Netherlands and 2Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, The Netherlands

The introduction of autologous stem cell transplantation the myeloma patients achieved a partial response (PR) or stable combined with the introduction of immunomodulatory drugs disease following rituximab therapy. All these patients expressed (IMiDs) and proteasome inhibitors has significantly improved CD20 on their myeloma cells.2 However, as only B15–20% of survival of multiple myeloma patients. However, ultimately the majority of patients will develop refractory disease, indicating all myeloma patients express CD20 on their bone marrow the need for new treatment modalities. In preclinical and clinical plasma cells, new targets for immunotherapy need to be studies, promising results have been obtained with several identified. The search for other targets has led to the develop- monoclonal antibodies (mAbs) targeting the myeloma tumor ment of mAbs targeting growth factor receptors or adhesion cell or the bone marrow microenvironment. The mechanisms molecules on myeloma cells. Other newly developed mAbs underlying the therapeutic efficacy of these mAbs include are directed against cellular or non-cellular components of the direct induction of tumor cell apoptosis via inhibition or activation of target molecules, complement-dependent cyto- bone marrow microenvironment, resulting in the neutraliza- toxicity and antibody-dependent cell-mediated cytotoxicity tion of growth factors, inhibition of angiogenesis, modulation (ADCC). The capability of IMiDs to enhance ADCC and the of mediators of bone disease and enhancement of the host modulation of various important signaling cascades in myelo- antitumor immune response (Figure 1 and Table 1). ma cells by both bortezomib and IMiDs forms the rationale to Besides direct effects via modulation of the activity of the combine these novel agents with mAbs as new treatment targeted antigen, mAbs can also induce tumor cell death strategies for myeloma patients. In this review, we will give an overview of various mAbs directly targeting myeloma tumor via complement-dependent cytotoxicity (CDC) and antibody- cells or indirectly via effects on the bone marrow micro- dependent cell-mediated cytotoxicity (ADCC) (Figure 2). The environment. Special focus will be on the combination of these process of ADCC is achieved through activation of Fc receptors mAbs with IMiDs or bortezomib. on myeloid and natural killer (NK) effector cells by tumor cell- Leukemia (2012) 26, 199–213; doi:10.1038/leu.2011.214; attached immunoglobins. Subsequent cytotoxicity is mediated published online 19 August 2011 through at least two different mechanisms: one involving the Keywords: multiple myeloma; immunotherapy; monoclonal antibody; immunomodulatory drugs; proteasome inhibitor; bone release of perforin and granzymes from effector cells, and the marrow micro-environment other involving death ligands Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). CDC is depen- dent on the interaction of the antibody Fc domains with the classic complement-activating protein C1q. There is also Introduction evidence that uptake of antibody-opsonized tumor cells and cellular fragments by antigen-presenting cells is associated with The introduction of autologous stem cell transplantation and enhanced antigen presentation, leading to tumor-specific T-cell 3 novel agents such as the proteasome inhibitor bortezomib and responses. the immunomodulatory drugs (IMiDs) thalidomide and lenali- The capability of IMiDs to activate multiple arms of the 4,5 domide has significantly improved survival of myeloma patient’s immune system including enhanced ADCC com- patients.1 However, the majority of myeloma patients ultimately bined with the modulation of various important signaling develop refractory disease, underscoring the need for new cascades in myeloma cells by both bortezomib and IMiDs therapeutic approaches. The search for new strategies has led forms the rationale to combine these novel agents with mAbs as not only to the development of second-generation immuno- new treatment strategies for myeloma patients. In this review, modulatory drugs (IMiDs) and proteasome inhibitors, but also we will give an overview of various classes of mAbs at different to compounds with different mechanisms of action, such stages of clinical development (Table 1). mAbs with similar as histone deacetylase inhibitors, Akt inhibitors and mTOR mechanisms of action are grouped together. Special focus will inhibitors. In addition, the successful application of immuno- be on mAbs that are currently being evaluated in clinical trials therapeutics such as the anti-CD20 monoclonal antibody and on combinations of these mAbs with IMiDs (Table 2) or (mAb) rituximab, the anti-CD52 mAb alemtuzumab and, most bortezomib (Table 3). recently, the anti-CD30–drug conjugate brentuximab vedotin in non-Hodgkin’s lymphoma, Hodgkin’s lymphoma and chronic lymphocytic leukemia has stimulated the development of mAbs targeting proteins involved in myeloma cell adhesion mAbs applicable for myeloma treatment. Interestingly, 32% of Myeloma cells interact with various cellular and non-cellular Correspondence: Dr NWCJ van de Donk, Department of Hematology, components of the bone marrow microenvironment, which University Medical Center Utrecht, Heidelberglaan 100, Utrecht promotes tumor growth, protects against antimyeloma agents, 3584CX, The Netherlands. E-mail: [email protected] leads to bone destruction and increases susceptibility to 6 Received 27 May 2011; accepted 5 July 2011; published online 19 infections by impairing the immune system. Adhesion is August 2011 mediated by cellular receptors and results in the activation of Monoclonal antibody-based therapy in myeloma NWCJ van de Donk et al 200 mAbs targeting mediators of NF-κB bone disease

RANKL↑, OPG↓ mAbs targeting α β β Notch-1 growth factors TNF- , TGF- , IL-1 , IL-6, IGF-I, Stromal cell and receptors bFGF, IL-6, VEGF VEGF, WNT

Raf NF-κB mAbs inhibting PI-3K JAK Adhesion MEK β-catenin Notch-1 adhesion molecules molecules Akt STAT3 P42/44 MAPK Anti-apoptosis Drug resistance Proliferation Growth factors VEGF, FGF-2, TNF-α, Proliferation Anti-apoptosis α Osteoclast TGF-, MMPs Drug resistance RANKL, MIP-1

Endothelial Myeloma cell cell VEGF, FGF-2, MMPs Fibronectine

mAbs targeting VEGF, IL-6, VEGF, IL-6, mAbs targeting growth factors TGF-β, IL-10 TGF-β, IL-10 mediators of and recetors bone disease

mAbs PD-1/PD-L1, KIR targeting VEGF, IL-6, growth factors DKK1, Bone resorption ↑ TGF-β, IL-10 mAbs and receptors activating sFRP-2 Bone formation ↓ NK cells Angiogenesis PD-1/PD-L1, CD200/CD200R mAbs B cell activating T cells

T cell NK cell Immune deficiency Osteoblast Dendritic cell

Figure 1 Targets of mAbs in multiple myeloma. Several mAbs that are currently being evaluated in clinical trials target important growth factor receptors and adhesion molecules present on myeloma cells. This will result in the inhibition of important signaling cascades and impair adhesion of myeloma cells to other cells in the bone marrow microenvironment. Other mAbs are directed against important cellular and non-cellular components of the microenvironment including growth factors, mediators of bone disease, T cells and NK cells. This results in neutralization of growth factors such as IL-6 or IGF-1, blockage of angiogenesis, inhibition of bone resorption, improvement of bone formation and enhancement of the host antitumor immune response.

important signaling cascades in both the myeloma cell and against myeloma cells.7,9 Furthermore, the combination of stromal cell, leading to protection against apoptosis and elotuzumab plus bortezomib resulted in improved antitumor increased production of several myeloma growth factors, activity in a mouse model.9 including interleukin-6 (IL-6), insulin growth factor-1 (IGF-1) Preliminary results from ongoing trials show encouraging and vascular endothelial growth factor (VEGF). Over the past results with acceptable toxicity for elotuzumab combination few years, various mAbs targeting adhesion molecules present therapy. No dose-limiting toxicity (DLT) was reported in a phase on myeloma cells showed great promise in both preclinical and 1 study evaluating elotuzumab 5, 10 or 20 mg on days 1, 8, 15 early clinical studies. and 22 of a 28-day cycle in combination with lenalidomide and dexamethasone. In this study of mostly lenalidomide-naı¨ve patients, at least PR was achieved in 82% of patients.10 In a CS1 phase 2 trial in lenalidomide-naı¨ve myeloma patients with a CS1 is a cell surface glycoprotein involved in myeloma cell median of two previous therapies, the combination of elotuzu- adhesion to bone marrow stromal cells.7,8 High expression of mab and lenalidomide plus dexamethasone was feasible with CS1 has been observed in multiple myeloma cell lines and in 85% at least PR.11 Two ongoing phase 3 trials will provide the majority of myeloma cells from patients (CS1 mRNA additional data to define the value of addition of elotuzumab to expressed by more than 97% of myeloma cells), whereas CS1 lenalidomide/dexamethasone in relapsed/refractory and newly is not detected at significant levels in normal tissues.7,8 diagnosed myeloma. The combination of elotuzumab with Elotuzumab (HuLuc63), a humanized mAb that binds to bortezomib was evaluated in a phase 1 study. Again no DLT CS1, inhibits myeloma cell adhesion to the bone marrow occurred, and at least PR was achieved in 48% of patients, stromal cells, which results in the inhibition of the stimu- including patients with bortezomib-refractory disease.12 Elotu- latory effects of bone marrow stromal cells on myeloma cell zumab infusion-related side effects included dizziness, nausea growth and survival.7,8 In addition, elotuzumab was found to and headache.11 induce myeloma cell death through ADCC.7,8 Elotutuzumab exerted significant antimyeloma activity in mouse models.7,8 Pretreatment of effector cells with lenalidomide significantly Syndecan-1 (CD138) increased elotuzumab-induced lysis of myeloma cells through Syndecan-1 (CD138) is a cell surface protein, which is stimulation of NK cell activity.7 Also, pretreatment of myeloma expressed on normal and malignant plasma cells.13 CD138 is cells with lenalidomide, bortezomib, dexamethasone or Akt not expressed by other hematopoietic cells, but expression has inhibitor perifosine enhanced elotuzumab-mediated ADCC been noted in epithelia of several organs and in renal tubules

Leukemia Monoclonal antibody-based therapy in myeloma NWCJ van de Donk et al 201 Table 1 mAbs under clinical evaluation in multiple myeloma and medullary region.14,15 It functions as a receptor for the extracellular matrix molecules collagen and fibronectin. CD138 Target Isotype also binds via its heparan sulfate chains to various soluble extracellular molecules, including hepatocyte growth factor and mAbs in advanced stages of clinical testing (phase 3) fibroblast growth factor.16 Elotuzumab (HuLuc63) CS1 Humanized IgG1 Siltuximab (CNTO 328) IL-6 Chimeric IgG1 Treatment of myeloma cell lines and myeloma cells from Denosumab RANKL Fully human IgG2 patients with the murine mAb B-B4 covalently linked to the potent antimicrotubule agent DM1 (B-B4-DM1) resulted in cell mAbs in earlier stages of clinical testing (phase 1 or 2) death, also when myeloma cells were adherent to bone marrow BT062 CD138 Chimeric IgG1 conjugated to DM4 stromal cells. The unconjugated B-B4 antibody alone had little 15 Lorvotuzumab (IMGN901) CD56 Humanized IgG1 activity. The immunoconjugate also suppressed tumor growth conjugated to DM1 in myeloma mouse models.15 The anti-CD138 antibody nBT062 Daratumumab CD38 Fully human IgG1 is a murine/human chimeric form of B-B4. Three immunocon- SAR650984 CD38 Humanized IgG1 BI-505 ICAM-1 Fully human IgG1 jugates of nBT062 with DM1 or DM4 were developed, which AVE1642 IGF-1R Humanized IgG1 also showed potent antimyeloma activities in vitro as well as in Figitumumab (CP-751,871) IGF-1R Fully human IgG2 vivo in myeloma mouse models.17 The most potent immuno- Bevacizumab (Avastin) VEGF Humanized IgG1 conjugate tested was nBT062-SPBD-DM4.17 In a dose-escala- LY2127399 BAFF Fully human IgG4 tion study, this immunoconjugate (BT062) was administered Milatuzumab CD74 Humanized IgG1 (hLL1, IMMU-115) once every 3 weeks in relapsed/refractory myeloma patients. Milatuzumab-DOX CD74 Humanized IgG1 DLT consisted of mucositis and the maximum-tolerated dose (hLL1-DOX; IMMU-110) conjugated (MTD) was defined as 160 mg/m2. Preliminary data from this to doxorubicin trial show evidence of clinical activity with disease stabilization Mapatumumab (HGS-ETR1) TRAIL-R1 Fully human IgG1 18 Dacetuzumab (SGN-40) CD40 (partially Humanized IgG1 in 13 out of 32 patients. On the basis of these data, a new agonistic) phase 1/2 study is initiated in which BT062 will be given in a Lucatumumab CD40 Fully human IgG1 more frequent dosing regimen. (HCD122; Chir-12.12) (antagonistic) The immunoconjugate nBT062-SPBD-DM4 also exerted Samalizumab (ALXN6000) CD200 Humanized IgG2/4 IPH2101 (1-7F9) KIR2DL1/2/3 Fully human IgG4 synergistic activity when combined with lenalidomide and 19 CT-011 PD-1 Humanized IgG1 bortezomib both in vitro and in mouse models, which forms BHQ880 DKK1 Fully human IgG1 the rationale for testing BTO62 in combination with novel MDX-1097 KMA Chimeric IgG1 agents in patients with relapsed/refractory myeloma. Abbreviations: BAFF, B-cell activating factor; DKK1, Dickkopf-1; ICAM-1, intercellular adhesion molecule-1; IGF-1R, insulin-like growth factor-1 receptor; IgG, immunoglobulin G; IL-6, interleukin-6; KMA, kappa myeloma antigen; mAb, monoclonal antibody; RANKL, receptor Neural cell adhesion molecule (CD56) activator of the NF-kB ligand; KIR, killer immunoglobulin-like receptor; Expression of CD56 (neural cell adhesion molecule) is found VEGF, vascular endothelial growth factor. on several cancers including the majority of myeloma cases.

Direct Effects

-Alterations in intracellular signaling -Inhibition of function of growth factor receptors -Inhibition of function of adhesion molecules -Neutralization of growth factors

Antibody-drug Conjugate

Monoclonal Antibody Growth Factor Bortezomib and/or Lenalidomide Antigen Signaling Cascades

CDC Fc Receptor Myeloma cell ADCC

C1q

NK cell Lenalidomide MAC Lysis

Cell Death

Figure 2 Mechanism of action of mAbs. mAbs have direct effects on the myeloma cell via modulation of the activity of the targeted antigen. Furthermore, CDC and ADCC may contribute to mAb-induced tumor cell death. The process of ADCC is achieved through activation of Fc receptors on myeloid and NK effector cells by tumor cell-attached immunoglobins. CDC is dependent on the interaction of the antibody Fc domains with the classic complement-activating protein C1q resulting in the accumulation of C3b, which acts as an opsonin and promotes phagocytosis. C3b also binds to C3 convertase to form a C5 convertase, leading to the induction of the membrane attack complex (MAC). In addition, antibody–drug conjugates have the potential of specifically delivering drugs to the myeloma cell. In clinical trials, mAbs are often combined with the novel agents lenalidomide and bortezomib. The preclinical rationale for these combinations is the enhancement of ADCC by lenalidomide and the modulation of various important signaling cascades by both lenalidomide and bortezomib.

Leukemia Monoclonal antibody-based therapy in myeloma NWCJ van de Donk et al 202 Table 2 Results from clinical studies evaluating a monoclonal antibody in combination with lenalidomide in relapsed/refractory multiple myeloma

Study Type of Regimen Schedule N Prior Response TTE Key toxicities study treatment

Agura Phase 1b Dacetuzumab- MTD not reached; 36 Median: 4 Evaluable Not Fatigue: 47% et al.100 len-dex highest dose level: Thal: NA patients reported Neutropenia: 28% Dacetuzumab Bort: NA (n ¼ 33) Thrombocytopenia: 25% 12 mg/kg once weekly Len: B50% XPR: 39% Diarrhea: 22% Len 25 mg on days VGPR: NA 1–21 of 28-day cycle CR: 3% Dex 40 mg on days 1, 8, 15 and 22 Callander Phase 2 Bevacizumab- Bevacizumab 10 mg/kg 31 Median: 3 Evaluable Not DVT: 10% et al.70 len-dex every 2 weeks Thal: NA patients reported Atrial fibrillation: 10% Len 25 mg on days Bort: NA (n ¼ 27) No hypertension 1–21 of 28-day cycle Len: 0% XPR: 70% or proteinuria Dex 40 mg on days 1, VGPR: NA 8, 15 and 22 CR: 15% Richardson Phase 2 Elotuzumab- Elotuzumab 10 or 59 Median: 2 Evaluable Not Infusion-related reactions: et al.11 len-dex 20 mg/kg on days 1, Thal: 53% patients reported dizziness (15%), nausea 8, 15 and 22 of Bort: 48% (n ¼ 26) (15%) and headache (9%) 28-day cycle in the Len: 0% XPR: 85% first 2 cycles, thereafter VGPR: 27% days 1 and 15 CR: 4% Len 25 mg on days 1–21 of 28-day cycle Dex 40 mg on days 1, 8, 15 and 22 Lonial Phase 1 Elotuzumab- MTD not reached; 29 Median: 3 Evaluable % without disease Two patients with severe et al.10 len-dex highest dose level: Thal: 59% patients progression after infusion reactions: one Elotuzumab 20 mg/kg Bort: 69% (n ¼ 28) 16 months: 53% with grade 4 hypersensitivity on days 1, 8, 15 and Len: 21% XPR: 82% reaction and one with 22 of 28-day cycle VGPR: NA two grade 3 stridor events in the first 2 cycles, CR: NA thereafter days 1 and 15 Len 25 mg on days 1–21 of 28-day cycle Dex 40 mg on day 1, 8, 15 and 22 Berdeja Phase 1 Lorvotuzumab- Lorvotuzumab on days 1, NA Median: NA Cohort 1 Not No drug-related et al. 26 len-dex 8 and 15 of 28-day cycle Thal: NA (n ¼ 3) reported grade 3 or 4 toxicities Len 25 mg on days 1–21 Bort: NA XPR: 67% of 28-day cycle Len: NA VGPR: 33.3% Dex 40 mg on days CR: 0% 1, 8, 15 and 22 Abbreviations: bort, bortezomib; CR, complete response; dex, dexamethasone; len, lenalidomide; MTD, maximum-tolerated dose; NA, not available; PR, partial response; thal, thalidomide; TTE, time to events; VGPR; very good partial response.

Normal plasma cells lack CD56 expression, whereas it is methasone in CD56-positive relapsed/refractory myeloma. In normally found on NK cells and a subset of T lymphocytes.20,21 the first dose level, two out of three myeloma patients achieved Lorvotuzumab (IMGN901) consists of a potent cytotoxic a response (one PR and one very good PR) without serious maytansinoid (DM1; a microtubule-disrupting agent) attached to adverse events.26 a CD56-binding mAb. Lorvotuzumab has potent activity as single agent against myeloma cell lines in the presence of stromal cells and in a myeloma mouse model.22 In combination CD38 with lenalidomide, synergistic activity was observed in vitro and CD38 is a type II transmembrane glycoprotein with ectoenzy- additive-to-synergistic efficacy was demonstrated in a myeloma matic activity involved in the production of calcium-mobilizing mouse model.23 Lorvotuzumab combined with bortezomib was compounds.27 Other functions ascribed to CD38 include found to be additive-to-synergistic against human myeloma receptor-mediated adhesion and signaling events.27 CD38 is xenografts in mice, whereas the combination in vitro showed highly and uniformly expressed on all myeloma cells. Under antagonism.23 normal conditions, CD38 is expressed at relatively low levels on A phase 1 study with CD56-positive relapsed/refractory mye- lymphoid or myeloid cells and in some tissues of non- loma patients showed that the MTD of lorvotuzumab was hematopoietic origin.27 Recently, a new human mAb directed 112 mg/m2, with DLT consisting of fatigue and renal failure. against CD38, daratumumab, was developed, which induced Overall lorvotuzumab was well tolerated and had antimyeloma myeloma cell killing through ADCC and CDC both in the activity (XMR: 18%).24,25 This trial and the preclinical studies presence or absence of bone marrow stromal cells.28,29 supported investigations of lorvotuzumab as part of a combina- In addition, daratumumab induced apoptosis when cross- tion regimen. An ongoing phase 1 study is evaluating the linked with anti-immunoglobulin G antibody.29 Also in mouse combination of lorvotuzumab with lenalidomide and dexa- models, potent antimyeloma activity for daratumumab was

Leukemia Monoclonal antibody-based therapy in myeloma NWCJ van de Donk et al 203 Table 3 Results from clinical studies evaluating a monoclonal antibody in combination with bortezomib in relapsed/refractory multiple myeloma

Study Type of Regimen Schedule N Prior Response TTE Key toxicities study treatment

Jakubowiak Phase 1 Elotuzumab- MTD not reached; 28 Median: 2 Evaluable Median TTP: SAE related to et al.12 bort-dex highest dose level: Thal: NA patients 9.5 months elotuzumab: one Elotuzumab 20 mg/kg Bort: 39% (n ¼ 27) grade 3 chest pain on days 1 and 11 Len: NA XPR: 48% and one grade 3 of a 21-day cycle VGPR: NA gastroenteritis Bort 1.3 mg/m2 on CR: 7% days 1, 4, 8 and 11 Dex in case of progression after cycle 2 or 3: 20 mg onday1,2,4,5,8,9, 11 and 12 Moreau Phase 1 AVE1642- MTD not reached; 11 Median: 4 XPR: 18% Not reported Majority of adverse et al.62 bortezomib highest dose level: Thal: NA VGPR: 0% events were related AVE1642 12 mg/kg on Bort: NA CR: 9% to bort day 1 of a 21-day cycle Len: NA Bort 1.3 mg/m2 on days 1, 4, 8 and 11 Belch Randomized Bort vs Bort 1.3 mg/m2 on 104 Median: NA XPR: 51% Median PFS: No evidence that et al. 110 phase 2 mapatumumab- days 1, 4, 8 and Thal: NA VGPR: NA 8.7 months mapatumumab bort 11 of a 21-day cycle Bort: 0% CR: 0% vs exacerbated toxicities vs Len: NA vs Median PFS: associated with bort Mapatumumab 10 XPR: 42% 4.7 and 5.7 or 20 mg/kg on day 1 VGPR: NA months for of a 21-day cycle CR: 3% 10 and 20 mg Bort 1.3 mg/m2 on mapatumumab, days 1, 4, 8 and 11 respectively Rossi Phase 2 CNTO CNTO 328 6 mg/kg every 21 Median: 2 Evaluable Median TTP: X3 grade et al.50 328-bort 2 weeks in 6-week cycle IMiDs: 38% patients 280 days neutropenia: 71% Bort 1.3 mg/m2 on days Bort: 0% (n ¼ 21) X3 grade 1, 4, 8 and 11 of a 21-day XPR: 57% thrombocytopenia: 38% cycle for the first four VGPR: 10% X3 grade infections: 24% 6-week cycles, thereafter CR: 14% four once-weekly doses in a 5-week cycle Dex in case of progression Abbreviations: bort, bortezomib; CR, complete response; dex, dexamethasone; IMiD, immunomodulatory drugs; len, lenalidomide; MTD, maximum- tolerated dose; NA, not available; PFS, progression-free survival; PR, partial response; thal, thalidomide; TTE, time to events; TTP, time to progression; VGPR; very good partial response. observed.28,30 Pretreatment of effector cells with lenalidomide increases after chemotherapy and high levels of ICAM-1 predict resulted in improved daratumumab-induced ADCC in myeloma poor response to therapy in chemo-naı¨ve patients.37 cell lines and primary myeloma cells.31,32 Furthermore, addition BI-505 is a fully human immunoglobulin G1 antibody specific of daratumumab to bortezomib or bortezomib plus lenalidomide for ICAM-1 that kills myeloma cells in vitro through induction of leads to a significant enhancement of myeloma cell killing.32,33 ADCC and apoptosis when crosslinked. In addition, BI-505 On the basis of these preclinical results, daratumumab is inhibits myeloma tumor growth in mice.38 On the basis of these currently evaluated in a phase 1/2 dose finding and safety study. data, a phase 1 dose-escalation study is currently recruiting Another anti-CD38 antibody is the humanized antibody relapsed/refractory myeloma patients to evaluate efficacy and SAR650984, which kills myeloma cells in vitro through direct toxicity of single-agent BI-505. pro-apoptotic activity, CDC and ADCC.34 In addition, in a mouse model it had strong antimyeloma activity as a single agent34,35 and synergistic activity when combined with mel- mAbs neutralizing growth factors or inhibiting growth- phalan.36 SAR650984 is being evaluated in a phase 1 dose- promoting receptors escalation trial to establish its MTD and DLT in patients with various types of CD38-positive hematological malignancies Bone marrow stromal cells, endothelial cells, osteoclasts and including myeloma. osteoblasts produce several myeloma growth factors, which after binding to their receptors lead to the activation of signaling pathways mediating growth, survival and resistance to antimye- ICAM-1 (CD54) loma drugs. Various mAbs have been developed that neutralize Intercellular adhesion molecule-1 (ICAM-1) mediates adhesion growth factors or inhibit the function of their receptors. of myeloma cells to bone marrow stromal cells and thereby contributes to cell adhesion-mediated drug resistance. ICAM-1 is highly expressed on the plasma cell surface in the majority of IL-6 and IL-6 receptor myeloma patients, but is also expressed on normal cell types, IL-6 is predominantly produced and secreted by bone marrow including endothelial cells, epithelial cells, fibroblasts and several stromal cells and is a major growth and survival factor for myeloma types of leukocytes.37 Expression of ICAM-1 on myeloma cells cells through activation of various signaling cascades, including

Leukemia Monoclonal antibody-based therapy in myeloma NWCJ van de Donk et al 204 JAK/STAT3, Ras/MAPK and PI3K/Akt.6 IL-6 also protects against plasma cells, whereas expression of this gene is observed in various antimyeloma agents including dexamethasone.6,39 myeloma cells from 31 to 50% of untreated patients.58 The Murine anti-IL-6mAbs have been used in the treatment of murine anti-IGF-1R mAbs mAVE1642(ref. 59) and a-IR3(ref. 57) myeloma patients, without major side effects and with some impair IGF-1R signaling and growth of myeloma cells. However, evidence of clinical activity.40,41 However, in a randomized the expected human anti-mouse antibody response limits the trial performed by the IFM group, addition of the murine anti-IL- use of these murine mAbs in the clinic, and resulted in the 6mAb, B-E8, to a tandem transplantation program had no effect generation of human or humanized anti-IGF-1R mAbs. on overall or event-free survival.42 In addition, rapid develop- The humanized anti-IGF-1R mAb AVE1642 inhibits growth ment of antibodies to mouse immunoglobulins results in rapid and strongly enhanced bortezomib-induced apoptosis in CD45- clearance and diminished efficacy of murine anti-IL-6mAbs.40,41 negative myeloma cell lines, but not in CD45-positive cell This led to the development of a chimeric mAb that lines.60 The mechanism of synergy between AVE1642 and neutralizes IL-6 (siltuximab; CNTO 328). Treatment of IL-6- bortezomib may be related to synergistic upregulation of Noxa, dependent cell lines with CNTO 328 resulted in a dose- which induces the release of the BH3-only activators that are dependent inhibition of proliferation both in the presence and responsible for Bax activation.60 On the basis of these results absence of bone marrow stromal cells.43 However, proliferation and phase 1 data, which showed good tolerability of AVE1642 of IL-6-independent cell lines was less influenced by treatment in advanced myeloma (except for grade 3 hyperglycemia in with CNTO 328.43 Clinical activity of this anti-IL-6mAb as diabetic patients),61 the combination of AVE1642 and bortezo- single agent in myeloma patients was limited,44,45 which mib was tested in relapsed/refractory myeloma. Although this resulted in the evaluation of combinations of CNTO 328 with combination was well tolerated, response rate was low with at other antimyeloma agents. least PR in only 18% of patients.62 Bortezomib activates various pro-apoptotic programs in Figitumumab (CP-751,871) is another fully human anti-IGF- myeloma cells, but may also lead to the activation of the 1R mAb in early clinical testing, which blocks IGF-1-induced antiapoptotic heat-shock protein response46 and accumulation signaling cascades and inhibits tumor growth both as single of the antiapoptotic protein Mcl-1.47 IL-6 is involved in the agent and more efficaciously in combination with chemother- transcriptional regulation of Mcl-1, heat-shock protein-70 and apeutic agents in several human tumor xenograft models.63 heat-shock protein-90,43 which formed the rationale of testing These results led to a phase 1 dose-escalation trial in relapsed/ CNTO 328 in combination with bortezomib. The cytotoxicity refractory myeloma patients with figitumumab as single agent, exerted by this combination in IL-6-dependent and -independent but with dexamethasone added when less than PR was myeloma cell lines was higher than the cytotoxicity of either achieved.64 No DLTs were identified. Monotherapy led to agent alone.43 Combined effects were associated with reduction disease stabilization in some patients, but objective responses of bortezomib-mediated induction of Mcl-1 and heat-shock were not observed. The addition of dexamethasone resulted in protein-70.43 CNTO 328 also increased the cytotoxicity of at least MR in 33% of the patients, including dexamethasone- dexamethasone in IL-6-dependent and -independent myeloma refractory patients.64 cell lines. Interestingly, addition of bortezomib to CNTO 328 Finally, promising preclinical results were obtained with the plus dexamethasone further enhanced the cytotoxicity of the fully human anti-IGF-1R antibody IMC-A12, which inhibits combination.48 Furthermore, blocking IL-6 signaling by CNTO tumor growth in mouse models as single agent and in 328 increased the antimyeloma effects of melphalan in a combination with melphalan and bortezomib.65 synergistic manner.49 On the basis of these preclinical data together with the acceptable safety profile of CNTO 328 monotherapy in myeloma,45 the combination of CNTO 328 Vascular endothelial growth factor with bortezomib was evaluated in a phase 2 trial with Myeloma cells and bone marrow stromal cells produce several bortezomib-naı¨ve relapsed/refractory myeloma patients with a pro-angiogenic factors such as VEGF, resulting in increased median of two prior lines of therapy. At least PR was achieved bone marrow angiogenesis contributing to myeloma growth and in 57% of the patients,50 which suggests a trend for a better survival. In addition to the antiangiogenic effects, VEGF also response rate than would have been expected with bortezomib functions as a growth factor for myeloma cells, stimulates IL-6 alone in relapsed/refractory myeloma.51 On the basis of these production in stromal cells, triggers myeloma cell migration and results, a randomized phase 3 study with bortezomib/dexa- inhibits maturation of dendritic cells. Altogether, this may methasone with CNTO 328 or placebo has started enrolling explain the poor survival in myeloma patients with elevated relapsed/refractory myeloma patients. There is also another levels of VEGF.66 Preclinical studies demonstrated that VEGF- randomized trial recruiting patients, which compares the neutralizing mAbs, such as the humanized mAb bevacizumab activity of CNTO 328 combined with VMP (bortezomib– (Avastin), inhibit activation of signaling cascades in myeloma melphalan–prednsione) to that of VMP alone in newly cells,67 reduce myeloma cell68 and endothelial cell prolifera- diagnosed myeloma patients. tion69 and inhibit capillarogenesis.69 Other agents inhibiting IL-6 signaling in preclinical stages of IMiDs also have antiangiogenic effects through downregula- development include the fully humanized anti-IL-6 mAb 133952 tion of factors such as VEGF, tumor necrosis factor-a (TNF-a) and and two humanized anti-IL-6 receptor mAbs, hPM153,54 and basic fibroblast growth factor. This formed the rationale to tocilizumab.55,56 evaluate the combination of bevacizumab, lenalidomide and dexamethasone in lenalidomide-naı¨ve relapsed/refractory mye- loma patients.70 At least PR was achieved in 70% of the patients, Insulin-like growth factor-1 receptor which is not statistically superior to lenalidomide-dexametha- IGF-1 is another important growth and survival factor for sone alone.71,72 In this study, achieving CR was associated with myeloma cells, through activation of PI3K/Akt and Ras/MAPK low plasma levels of macrophage inflammatory protein-1a and signaling pathways.6 Importantly, IGF-1 also protects myeloma VEGF.70 In the same way, the combination of thalidomide with cells against anticancer drugs including bortezomib.57 IGF-1 bevacizumab yielded similar results when compared to single- receptor (IGF-1R) gene is not expressed by normal B cells or agent thalidomide.73 Another ongoing randomized controlled

Leukemia Monoclonal antibody-based therapy in myeloma NWCJ van de Donk et al 205 phase 2 study is currently evaluating the combination of CD40L interaction, myeloma proliferation and migration are bevacizumab and bortezomib versus bortezomib and placebo. induced via the PI3K/AKT/NF-kB signaling pathway.93 CD40 This study is based on in vitro additive antimyeloma activity stimulation is also important for adhesion of myeloma cells to between bortezomib and VEGF small interfering RNA silencing.68 bone marrow stromal cells, leading to increased production of IL-6 and VEGF in both cell populations.92,94,95 Dacetuzumab (SGN-40) is a humanized partially agonistic B-cell activating factor anti-CD40 antibody, which induces ADCC against CD40- B-cell activating factor (BAFF) is a TNF superfamily member and postive myeloma cells.96 Although dacetuzumab does not produced in the bone marrow microenvironment by monocytes, block CD40L-induced signaling, it upregulates cytotoxic ligands osteoclasts and neutrophils.74 Binding of BAFF to the three such as Fas ligand and TRAIL, and downregulates expres- different TNF-R-related receptors, TACI, BCMA and BAFF-R, sion of the IL-6 receptor, resulting in synergistic killing of triggers activation of NF-kB, PI3K and MAPK pathways, resulting myeloma cells in combination with the protein synthesis in survival and dexamethasone resistance of myeloma cells.75,76 inhibitor cycloheximide.97 Also, addition of lenalidomide to BAFF also promotes adhesion of myeloma cells to bone marrow dacetuzumab enhanced cytotoxicity against myeloma cells. stromal cells.77 In a mouse model, an anti-BAFF-neutralizing This was mediated via both direct antiproliferative and apoptotic antibody had antimyeloma activity and inhibited osteoclasto- effects, as well as increased sensitivity of myeloma cells to genesis.75 On the basis of these results, a phase 1 study is ADCC by NK effector cells.98 currently enrolling relapsed/refractory myeloma patients to In a phase 1 dose-finding study with relapsed/refractory evaluate the combination of the neutralizing anti-BAFF anti- myeloma patients (median 5 lines of prior therapy), the MTD body, LY2127399, combined with bortezomib. of dacetuzumab as single agent was 12 mg/kg per week with intrapatient dose escalation to prevent cytokine release syndrome.99 Although dacetuzumab as a single agent was CD74 generally well tolerated, no objective clinical responses were CD74 (invariant chain, Ii) associates with the major histocom- reported.99 On the basis of the promising in vitro findings, patibility class IIa and b chains and directs the transport of these the combination of dacetuzumab with lenalidomide and dexa- complexes to endosomes and lysosomes.78,79 CD74 also func- methasone was evaluated in a phase 1b dose-escalation study tions as a receptor for macrophage migration-inhibitory factor.80 in patients with relapsed/refractory myeloma with a median Binding of macrophage migration-inhibitory factor to CD74 of 4 prior treatments. The combination was well tolerated leads to proliferation and survival of cells through activation of and the MTD was not reached. At least PR was achieved signaling cascades including the NF-kB pathway.81–83 CD74 is in 39% of patients, including responses in lenalidomide- expressed on the majority of myeloma cells in B90% of the exposed patients.100 Another dose-escalation study is evaluating patients.84 In addition to its expression on antigen-presenting dacetuzumab and bortezomib in the setting of relapsed/ cells, it has restricted expression in normal human tissues.84 refractory disease. Crosslinked milatuzumab (hLL1, IMMU-115), a humanized XmAbCD40 is another humanized mildly agonistic anti- anti-CD74 mAb, blocks CD74 and thereby prevents NF-kB CD40 antibody with mutations in its Fc domain leading to activation, resulting in growth-inhibitory effects and induction of increased FcgR binding. This Fc-engineered anti-CD40mAb had apoptosis in myeloma cell lines.85,86 Milatuzumab lacks CDC or markedly increased ADCC activity against myeloma or lym- ADCC activity.86 In a myeloma mouse model, treatment with phoma cells, when compared to the native immunoglobulin G1 milatuzumab resulted in significant survival extensions without analog. This translated into a higher efficacy of XmAbCD40 to the need for an exogenous crosslinking agent.85,86 Doxorubicin inhibit tumor growth in mice.101 conjugated to this antibody (IMMU-110) had cytotoxic effects Finally, there is a fully human antagonistic anti-CD40 mAb on myeloma cells in vitro and inhibited tumor growth in a lucatumumab (HCD122; CHIR-12.12), which blocks CD40L- mouse model without significant toxicity.87 Also the immuno- mediated signaling, inhibits CD40L-induced IL-6 and VEGF toxin, 2L-Rap-hLL1-g4P, composed of frog RNase fused to the secretion in myeloma and stromal cells, and reduces CD40L- anti-CD74 antibody killed myeloma cells in vitro.88 triggered myeloma cell adhesion to fibronectin or stromal cells. Interestingly, crosslinked milatuzumab enhances the efficacy This results in the inhibition of CD40L-induced growth of of bortezomib, doxorubicin or dexamethasone in vitro.85 myeloma cells in the presence or absence of stromal cells.102 Furthermore, in a myeloma mouse model combining milatuzu- In addition, lucatumumab is a potent mediator of ADCC.102 mab and bortezomib increased survival significantly compared Preliminary data from a phase 1 study in patients with relapsed/ with either treatment alone.85 The observation that in a subset of refractory myeloma showed that lucatumumab as a single agent myeloma patients constitutive NF-kB activity is refractory to was well tolerated, with one out of nine patients achieving inhibition by bortezomib89 may be the explanation for the PR and two with stable disease. The MTD was not (yet) reached synergism between these two differentially acting NF-kB and grade 1–2 toxicities mainly consisted of chills, nausea, inhibitors. In a phase 1 trial, milatuzumab as single agent pyrexia and arthralgia, which were most often reported during showed no severe adverse effects in relapsed/refractory myelo- the first infusion.103 ma patients and it stabilized the disease in some patients for up to 12 weeks.90 The doxorubicin conjugate of milatuzumab is currently being evaluated in relapsed/refractory myeloma. mAbs activating death receptors

The TNF family members Fas ligand and TRAIL induce apoptosis CD40 upon binding to the death receptors Fas, and TRAIL receptor 1 CD40 is a member of the TNF receptor family and normally (TRAIL-R1; DR4) or TRAIL receptor 2 (TRAIL-R2; DR5), expressed on B cells, dendritic cells, several types of epithelial respectively.104,105 TRAIL also binds to the decoy receptors cells and endothelial cells. In addition, it has a relatively high DcR1 and DcR2 and to the soluble decoy receptor osteopro- expression on the majority of myeloma cells.91,92 Upon CD40– tegerin (OPG), which is produced by osteoblasts and stromal

Leukemia Monoclonal antibody-based therapy in myeloma NWCJ van de Donk et al 206 cells. In this section, we will discuss TRAIL receptor antibodies, receptors recognize essentially all HLA-C allotypes.117 Cyto- which have been evaluated in myeloma patients. toxicity of both purified NK cells and isolated peripheral blood mononuclear cells against myeloma cell lines was enhanced by 1-7F9.118 Combination therapy of lenalidomide, which also TRAIL-R1 and -R2 activates NK cells, with 1-7F9 resulted in increased NK cell Antimyeloma activity was observed for the fully human cytotoxicity directed towards the myeloma cells when com- agonistic antibody directed against TRAIL-R1 (mapatumumab, pared to treatment with either agent alone.118 Potential HGS-ETR1) and to a lesser extent for the anti-TRAIL-R2 antibody mechanisms for the enhancement of NK cell activity against (lexatumumab, HGS-ETR2).106 Importantly, the antimyeloma myeloma cells by the combination of lenalidomide and 1-7F9 effect of anti-DR4 and anti-DR5 antibodies was not affected by include increased NK cell migration to myeloma cells, the presence of cells of the bone marrow microenvironment, upregulation of several NK cell activating ligands and increased whereas these cells protected myeloma cells against TRAIL- interferon-g and granzyme B production by the NK cells.119 induced apoptosis.107 This protective effect was at least partly Preliminary results from a dose-escalation study in patients mediated by OPG.107 with relapsed/refractory myeloma showed that 1-7F9/IPH2101 Bortezomib treatment is associated with the upregulation of as a single agent is well tolerated. Objective responses were not TRAIL and its receptors in B-CLL and lymphoma cells.108 observed; however, some patients achieved stabilization of Furthermore, blockage of TRAIL-R1 and -R2 expression using disease.120 Antitumor activity and toxicity of single-agent 1-7F9 RNA interference, which prevents TRAIL apoptotic signaling, will also be determined in smoldering myeloma patients and in inhibited proteasome inhibitor-induced apoptosis.108 Indeed, patients who achieved a stable PR or very good PR following combined bortezomib plus mapatumumab treatment resulted in first-line therapy. The hypothesized synergistic activity of enhanced myeloma cell killing when compared to either agent lenalidomide and 1-7F9 is currently being evaluated in a phase alone, in both wild-type and bortezomib-resistant cells.109 In 1/2 trial with relapsed/refractory myeloma patients. contrast with the preclinical data, a randomized phase 2 study in relapsed/refractory myeloma showed that addition of mapatu- mumab to bortezomib treatment did not increase response rate PD-L1 or progression-free survival compared to bortezomib alone.110 Another way of improving antitumor activity of NK cells against There was no evidence that the anti-TRAIL-R1 antibody myeloma cells is by modulating the PD-1/PD-L1 axis, which exacerbated the toxicities associated with bortezomib.110 downregulates the immune response.121 PD-1 is not present on NK cells from healthy donors, but can be upregulated by exogenous IL-2.113 In contrast, PD-1 is present on NK cells from mAbs improving the antitumor immune response patients and interaction with PD-L1 on myeloma cells inhibits NK cell function.113 PD-L1 is expressed on plasma cells from the NK cells are not only important effectors of ADCC, but also majority of myeloma patients, but only rarely on plasma cells capable of killing myeloma cells in an antibody-independent from MGUS patients and not on normal plasma cells.122 CT-011 manner.111 NK cell effector function is regulated by multiple is a humanized anti-PD-1 mAb, which enhanced NK cell types of activating and inhibitory receptors that recognize function against myeloma tumor cells through increased NK cell ligands expressed on potential target cells.112 NK cell cytotoxi- trafficking, improved immune complex formation between city is incited when a target cell abundantly expresses ligands myeloma cells and NK cells, and increased NK cell cytotoxicity. for activating receptors while few or no major histocompatibility Lenalidomide not only activates NK cells, but also down- class I ligands for inhibitory receptors, such as the killer regulates PD-L1 expression on myeloma cells and synergized immunoglobulin-like receptors (KIR), are expressed. Impor- with CT-011 in the activation of NK cells and subsequent killing tantly, this NK versus myeloma effect diminishes during disease of myeloma cells.113 Apart from effects on NK cells, blockade of progression through various mechanisms including upregulation the PD-1/PD-L1 interaction with anti-PD-L1 mAbs enhanced of inhibitory ligands such as PD-L1113 and shedding of soluble myeloma-specific T-cell immunity in vitro and in vivo.122,123 forms of activating ligands like major histocompatibility complex Furthermore, CT-011 also enhanced T-cell responses to auto- class I-related chain A.114 Various approaches including logous dendritic cell/myeloma fusion vaccines.124 This may be IMiDs,111,115 mAbs and cytokines such as IL-2116 target the NK explained by increased expression of PD-1 on T cells from cell to improve its ability to induce myeloma cell death. myeloma patients when compared to healthy controls. In addition, suppression of myeloma-specific cytotoxic T cells On the basis of these data, a phase 1 clinical trial was initiated by cytokines, such as TGF-b, recruitment of regulatory T cells to study the effect of CT-011 in patients with advanced hemato- and altered expression of immune suppressor molecules on logical malignancies including myeloma patients. CT-011 was myeloma cells or immune effector cells, also contributes to demonstrated to be well tolerated with evidence of antitumor immune evasion in myeloma. Furthermore, the function of activity. Treatment with CT-011 was accompanied with an dendritic cells is severely impaired in myeloma, including elevated percentage of peripheral blood CD4-positive T cells.125 reduced expression of co-stimulatory molecules.4 Here, we will The combination of CT-011 with an IMiD may further enhance review mAbs that are currently evaluated in myeloma patients, host antitumor immunity and warrants further investigation. which reverse resistance to immune-mediated destruction of Alternatively, the immune response to myeloma may be myeloma cells. enhanced by anti-PD-L1 antibodies such as MDX-1105.

KIR CD200 A fully human anti-KIR antibody directed against KIR2DL1/2/3 CD200 is expressed on B and T lymphocytes, endothelial cells, was developed (1-7F9 or IPH2101), which blocks the interac- dendritic cells and neurons, but normal plasma cells lack tions with their HLA-C ligands and thereby enhances NK cell CD200 expression.126 However, the majority of myeloma activity. Collectively, the inhibitory KIR2DL1, -2 and -3 patients have CD200 expression on their tumor cells, which

Leukemia Monoclonal antibody-based therapy in myeloma NWCJ van de Donk et al 207 correlated with shorter survival after autologous stem cell inhibited tumor growth both in vitro and in mouse models.140 In transplantation,126,127 possibly explained by the CD200- the 5T2MM mouse model, BHQ880 prevented the development mediated suppression of the cytotoxic T-cell immune response of osteolytic bone lesions.141 On the basis of these preclinical leading to tumor immune evasion. studies, a phase 1/2 study was initiated in which relapsed/ Samalizumab (ALXN6000) is a humanized immunoglobulin refractory myeloma patients received BHQ880 in combination G2/4 anti-CD200 antibody, which blocks CD200–CD200R with zoledronic acid. This combination was well tolerated with interactions without ADCC or CDC activity. In mouse models, increases in bone mineral density in several patients.142 this antibody improved peripheral blood mononuclear cell- Combination of denosumab or BHQ880 with bortezomib mediated tumor growth inhibition in CD200-positive tumors.128 would be an interesting approach, as bortezomib not only In a phase 1/2 study, single-agent samalizumab was evaluated inhibits bone resorption, but also increases bone forma- in patients with myeloma or chronic lymphocytic leukemia. tion.143,144 Promising results from preclinical studies showed Treatment was well tolerated with antitumor activity evident in that mAbs targeting activin A,145 sFRP-2146 and macrophage some patients receiving multiple cycles, which was associated inflammatory protein-1a147,148 hold promise as new treatment with reduction in regulatory T cells and increase in activated modalities for myeloma bone disease. T cells.129

Concluding remarks and future perspectives mAbs targeting mediators of bone disease Although survival of myeloma patients has significantly in- More than half of the myeloma patients present with bone creased, almost all patients will develop refractory disease. disease at diagnosis. Increased osteoclastogenesis and osteo- Median overall survival in patients who relapse after or are clast activation combined with reduced osteoblastogenesis and refractory to bortezomib and either thalidomide or lenalidomide impairment of osteoblast function result in the development of is only 6 months,149 indicating the need for new treatment osteolytic bone lesions. Importantly, osteoclast activity may also strategies. At this moment, encouraging preclinical results as contribute to myeloma cell growth and survival,130 whereas well as clinical data indicate that mAbs targeting growth factor osteoblasts may inhibit myeloma cell growth.131,132 Bisphos- receptors and adhesion molecules present on myeloma cells, or phonates impair osteoclast activity and thereby reduce (but not components in their surrounding microenvironment, such as completely prevent) skeletal complications. However, bisphos- growth factors, pro-angiogenic factors, mediators of myeloma phonates are unable to induce bone formation and repair of bone disease and immune effector cells, are emerging as lytic lesions. Therefore, new treatment approaches are needed. important antimyeloma agents. Multiple mediators that lead to the severe imbalance between In this review, we grouped together mAbs with similar increased osteoclastic bone resorption and decreased bone mechanisms of action. However, as several mAbs have multiple formation have been identified, leading to the development effects on the tumor and microenvironment, these antibodies of several mAbs for the treatment of myeloma bone disease. often belong to more than one class. For example, antibodies Two of these mAbs, denosumab and BHQ880, are in clinical inhbiting VEGF inhibit tumor growth, impair angiogenesis, and development. given the immunesuppressive effects of VEGF may also improve Receptor activator of the NF-kB ligand (RANKL) is present on immune function. myeloma cells and can be induced on stromal cells by myeloma An important observation from these clinical trials is that cells. Interaction of RANKL with RANK leads to increased mAbs alone have limited antitumor activity; however, combina- osteoclastogenesis and activation of osteoclasts. OPG inhibits tion with other antimyeloma agents is a promising new RANK/RANKL interactions and myeloma cells reduce its treatment strategy in myeloma. Of great interest is the production by osteoblasts and stromal cells. The critical role combination of various therapeutic antibodies with an IMiD of the OPG/RANKL/RANK system for the development of such as lenalidomide, which not only has potent direct myeloma bone has been demonstrated by using recombinant antimyeloma activity, but also significantly enhances ADCC. OPG, RANK-Fc or anti-RANKL antibodies. These preclinical In addition, encouraging data are available showing that the studies not only demonstrated inhibition of skeletal destruction, combination of a proteasome inhibitor with a mAb induces but also decrease in tumor burden.130,133–135 In a phase 2 trial myeloma cell death in a synergistic way, through independently with myeloma patients, denosumab, which is a fully human targeting the same pathway or via inhibition of different neutralizing anti-RANKL mAb, suppressed the RANKL pathway signaling cascades. In this respect, several mAbs are currently as evidenced by reductions in bone turnover markers. Denosu- being evaluated in phase 2 trials in combination with other mab was well tolerated, but no objective antitumor responses antimyeloma therapies to determine the best partner drugs for were observed.136 Recently, a phase 3 study demonstrated that antibody-based immunotherapy in terms of disease response denosumab was non-inferior to zoledronic acid in preventing and toxicity. On the other hand, some mAbs including or delaying skeletal-related events in patients with solid tumors elotuzumab and siltuximab, which showed promise in phase 2 or myeloma. Patients treated with denosumab experienced trials, have proceeded to randomized controlled phase 3 trials to a greater suppression of bone turnover markers than with define their value as adjunctive treatment to novel agents in zoledronic acid.137 improving survival (Table 1). Furthermore, an increased under- Production of the soluble Wnt inhibitor Dickkopf-1 by standing of unique properties of myeloma cells and their myeloma cells inhibits differentiation of osteoblast precursor environment will probably lead to the design of more rational cells,138 which explains the association between high Dickkopf-1 antibody-containing combination therapies that will not only levels and the presence of osteolytic bone lesions.139 The fully target important signaling cascades in the myeloma cells, human Dickkopf-1-neutralizing antibody BHQ880 reversed but also important extrinsic components that contribute to the inhibitory effect of myeloma cells on osteoblastogenesis, myeloma cell survival, such as adhesion of tumor cells to stimulated bone formation, inhibited stromal cell/myeloma cell stromal cells, pro-survival signals in the microenvironment, adhesion, reduced production of IL-6 by stromal cells and mechanisms of immune escape and mediators of bone disease.

Leukemia Monoclonal antibody-based therapy in myeloma NWCJ van de Donk et al 208 Such combinations will result in more effective eradication of the identification of subgroups of myeloma patients that will likely tumor, while at the same time drug resistance will be prevented. benefit from a certain antibody-based combination therapy. A second important conclusion from this review is that Future studies should incorporate analysis of biomarker compo- although expression of mAb targets is not always restricted to the nents that can predict the safety and effectiveness of new mAbs. myeloma tumor cells, toxicity of the mAbs is relatively mild. On Increased understanding of the biology of myeloma including the other hand, minimizing side effects of chemotherapy can the importance of the cross-talk between tumor cells and the also be achieved by using myeloma-specific antibody con- microenvironment has resulted in the development of several jugates to specifically deliver cytotoxic drugs to the tumor cells. novel therapeutic strategies including several classes of mAbs, This approach will allow the administration of lower doses of which specifically target the myeloma tumor cell or various cytotoxic drugs and may therefore reduce toxicity of antimye- components of the bone marrow microenvironment. Altogether, loma therapies. In myeloma, promising results from early the introduction of novel antimyeloma agents combined with clinical testing have been obtained for several antibody drug the identification of biomarkers that are predictive of therapeutic conjugates (Table 1). responses will eventually result in a more individualized In this review, we mainly focused on mAbs that are targeted therapy, which will hopefully be better tolerated and currently under clinical evaluation or have been tested in more effective, resulting in further improvement of survival of clinical trials. However, multiple other mAbs are in various myeloma patients. stages of preclinical development, including antibodies target- ing FGFR3,150–152 HM1.24/CD317,153,154 b2-microglobulin,155 a4-integrin (CD49d; subunit of VLA-4)156 and PSGL-1 (P- Conflict of interest selectin glycoprotein ligand-1; CD162).157 Other promising F agents include MDX-1097, which targets kappa light chains not ND: research funding/contracted research Celgene. HL: research F associated with heavy chains present on the surface of kappa- funding/contracted research Celgene, Genmab and Ortho- positive myeloma cells (kappa myeloma antigen), and is Biotech. SK and TM: none. currently evaluated in a phase 2 trial based on promising preclinical data158 and favourable phase 1 safety data;159 Acknowledgements antibody–drug conjugates binding to FcRL5;160 an anti-IL-17A antibody that inhibits myeloma cell growth and osteoclast 161 We thank Dr Constantine S Mitsiades (Dana-Farber Cancer development; and a bispecific antibody that neutralizes both Institute, Harvard Medical School, Boston, MA, USA) for useful VEGF and angiopoietin-2, resulting in strong inhibition of discussions and comments on the manuscript. angiogenesis.162 However, despite the enormous progress that has been made, many challenges still remain such as the unravelling of mechanisms References of therapeutic resistance to mAbs. 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