Monoclonal antibodies: potential new therapeutic treatment against multiple myeloma

REVIEW ARTICLE

Monoclonal antibodies: potential new therapeutic treatmentagainst multiple myelomaAlessandro Allegra, Giuseppa Penna, Andrea Alonci, Sabina Russo, Bruna Greve, Vanessa Innao,Viviana Minardi, Caterina Musolino

Division of Haematology, University of Messina, Messina, Italy

Abstract

Despite recent treatments, such as bortezomib, thalidomide, and lenalidomide, therapy of multiple

myeloma (MM) is limited, and MM remains an incurable disease associated with high mortality. The

outcome of patients treated with cytotoxic therapy has not been satisfactory. Therefore, new therapies

are needed for relapsed MM. A new anticancer strategy is the use of monoclonal antibodies (MoAbs) that

represent the best available combination of tumor cytotoxicity, environmental signal privation, and immune

system redirection. Clinical results in patients with relapsed/refractory MM suggest that MoAbs are likely

to operate synergistically with traditional therapies (dexamethasone), immune modulators (thalidomide,

lenalidomide), and other novel therapies (bortezomib); in addition, MoAbs have shown the ability to

overcome resistance to these therapies. It remains to be defined how MoAb therapy can most fruitfully

be incorporated into the current therapeutic paradigms that have achieved significant survival earnings in

patients with MM. This will require careful consideration of the optimal sequence of treatments and their

clinical position as either short-term induction therapy, frontline therapy in patients ineligible for ASCT, or

long-term maintenance treatment.

Key words multiple myeloma; monoclonal antibodies; therapy; microenvironmental; tumor cytotoxicity; immune system

Correspondence Prof. Caterina Musolino, Division of Haematology, University of Messina, Via Consolare Valeria 98124 Messina,

Italy. Tel: +39 090 221 2364; Fax: +39 090 2212355; e-mail: [email protected]

Accepted for publication 13 March 2013 doi:10.1111/ejh.12107

General considerations on monoclonal

antibodies and multiple myeloma

Multiple myeloma (MM) is a progressive and fatal diseasecharacterized by the malignant proliferation of plasma cellsin the bone marrow and overproduction of monoclonalimmunoglobulin or light-chain proteins. MM therapy remainschallenging. Despite recent therapies, such as bortezomib,thalidomide, and lenalidomide, treatment is limited and palli-ative, and MM remains an incurable disease associated witha median survival of 4 yr (1).Patients refractory to both bortezomib and either lenalido-

mide or thalidomide were found to have a median overall sur-vival of 9 months (2). Then, the outcome of patients with MMtreated with cytotoxic therapy has not been adequate, and newtreatments are needed for relapsed or refractory MM.A new anticancer strategy is the use of monoclonal anti-

bodies (MoAb) that operate through completely different

mechanisms of action. MoAbs directed against MM-associated markers should be taken into account in clinicalpractice, because they could represent the best availablecombination of tumor cytotoxicity, environmental signal pri-vation, and immune system redirection (3).Ideally, targets for therapeutic MoAbs should be specifi-

cally expressed on cancerous cells but not on normal cells. Infact, monoclonal antibodies are an interesting thera-peuticoption in MM because they are specific to a tumor-associatedtarget and have been successfully employed in the treatmentof patients with other hematologic diseases (4).Moreover, given that the mechanisms of cytotoxicity by MoAb

therapy are quite different from those of chemotherapeutic drugs,MoAb therapy can work synergistically with chemotherapy.In MM, MoAb can be directed against a large variety of anti-

gen targets, which can be expressed either on myeloma cells oron components of the bone marrow microenvironment [bonemarrow stromal cells (BMSCs) or signaling molecules] (3, 5).

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 441

European Journal of Haematology 90 (441–468)

Therapeutic MoAbs use one or more mechanisms toreduce tumor burden in patients. They could directly induceapoptosis or growth arrest upon binding to cell surface anti-gen on tumor cells. Rituximab and mapatumumab couldcause growth inhibition or apoptosis signaling to blocktumor cell growth and survival. Such mechanism of actionwas employed by MoAbs conjugated with toxins, that is,maytansinoids for anti-CD56 and anti-CD138, thus directlytarget and eliminate tumor cells.Most of the approved therapeutic MoAbs belong to IgG1

subclass, which has a long half-life, and activate immuneeffector functions. After the binding of MoAbs to a specifictarget on a tumor cells, antibody-dependent cellular cytotoxicity(ADCC) is triggered by interactions between the Fc region ofan antibody bound to a tumor cell and Fc receptors,particularly FcRI and FcRIII, on immune effector cells such asnatural killer (NK) cells, macrophages, and neutrophils.MoAb-coated tumor cells are phagocytosed by macrophagesor undergo cytolysis by NK cells. In the case of complement-dependent cytotoxicity (CDC), recruitment of C1q by IgGbound to the tumor cell surface is the first step. This triggers aproteolytic cascade that induces production of the effectormolecule, C3b, and then formation of a membrane attack com-plex that kills the target cell by disrupting its cell membrane.MoAbs have been additionally designed to functionally

block both autocrine- and paracrine-secreted cytokines andgrowth factors as well as molecules mediating MM–stromalcell interaction.Although expression of MoAb targets is not always con-

fined to the myeloma cells, toxicity of the MoAbs is rela-tively mild. On the other hand, minimizing side effects ofchemotherapy can also be achieved using myeloma-specificantibody conjugates to specifically deliver cytotoxic drugs tothe tumor cells. This method will consent the administrationof lower doses of drugs and may therefore reduce toxicity ofantimyeloma treatments (6, 7).A vast assortment of antigens may be targeted in MM

treatments, including those implicated in cell survival, anti-apoptotic pathways, angiogenesis, and interactions betweenMM cells and BMSCs (8).These potential targets include mediators of adhesion, sig-

naling molecules, cell surface receptors, and plasma cellgrowth factors.

MoAbs Targeting tumor cells

Several MoAbs directed against MM cell surface are beinginvestigated as potential targets in MM patients (Table 1).

Anti-CD20 MoAb

Several works have shown that MM includes clonotypic Blineage cells at stages earlier than the compartment of malig-nant plasma cells in the bone marrow (9), and the circulating

component of the MM clone includes at least two distinctCD19+ CD20+ B-cell compartments as well as CD138+

CD20+ plasma cells. Pilarski et al. evaluated them before,during, and after treatment of patients with rituximab(anti-CD20), followed by quantifying B-cell subsets over a5-month period during and after treatment. Overall, all threetypes of circulating B lineage cells persist despite treatmentwith rituximab. The inability of rituximab to prolong sur-vival in MM may result from this failure to deplete CD20+

B and plasma cells in MM (10).Indeed, previous studies demonstrated only minimal activ-

ity of anti-CD20 rituximab and antibodies against plasmacell-specific CD38 antibodies in MM (11–13). However,

Table 1 Monoclonal antibodies (MoAbs) targeting tumor cells

Target Name

CD20 Rituximab

Tositumomab

20-C2-2b Veltuzumab

CS1 Elotuzumab

CD138 B-B4

BC/B-B4

DL-101

1 D4

1.BB.210

MI15

2Q1484

5F7

104-9

281-2

nBT062-SMCL-DM1

nBT062-SPDM4

nBT062-SPP-DM1

CD38 Doratumumab

MOR202

CD40 Lucatuzumab

Lorvotuzumab

IGF-1 AVE1642

AMG479 IMCA12

R15507

Figitumumab

Dalotuzumab

HMI.24 (CD317) AHM

Defucosylated AHM

XmAb 5592

CD48 Anti-CD48 MoAb

b2 m IgG anti-b2 m

IgM anti-b2 m

CD70 SGN-70

CD74 Milatuzumab

HLADR ID09C3

2D7-DB

CD229 Anti-CD229

GM2 ganglioside BIW-8962

CD54 (ICAM-1) BI-505

Ku 5E2

442 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Monoclonal antibodies and multiple myeloma Allegra et al.

although the CD-2 subgroup of myeloma frequently overex-presses CD20 and few studies trying to prove otherwise(14), clinical studies of rituximab treatment in MM have forthe most part been discouraging, with few patients achievingonly minimal responses (15). Results from a clinical phase 2trial in relapsed MM showed that rituximab treatmentyielded significant reduction in circulating B cells but hadno beneficial clinical effect (16).Moreover, rituximab was tested for maintenance therapy

in MM following autologous hematopoietic stem cell trans-plantation (SCT) (12). The use of rituximab in this sort ofpatients was associated with an unexpectedly high rate ofearly relapse. The authors then hypothesized a possible rolefor rituximab in provoking an additional reduction in thenormal, residual B-cell activity.MM is generally not considered as a disease adequate for

anti-CD20 therapy due to weak and various expression ofCD20 in the preponderance of subjects. In contrast, otherstudies demonstrated that the CD20+ phenotype is associatedwith patients with t(11,14)(q13;q32) and with shorter sur-vival (17) and that sporadic responses have been achieved inpatients with CD20+ myelomatous plasma cells (18, 19).The failing of rituximab is probably attributable to the

small number of MM subjects (estimated at 13–22%) whoexpress CD20 in plasma cells, but a different mechanism thatmay render MM refractory to rituximab is the possibility thatMM cells express increased levels of complement-inhibitingproteins, such as the presence of the complement regulatorCD59 on myeloma makes complement-mediated cytotoxicityinefficacious (16, 20–22). In addition, Fc-c receptor polymor-phism may limit the efficacy of ADCC as a killing mecha-nism. Finally, the administration of rituximab in MM maycause a selective loss of CD20 expression.In spite of this, recently, a different anti-CD20 antibody

was used in MM patient. I-131 tositumomab is a radiola-beled murine anti-CD20 antibody, which is highly effectivein the treatment of low-grade B-cell NHL. It was tested in asingle arm, phase 2 study. With a median follow-up of 4 yr,none of the responding patients have progressed. Then,I-131 tositumomab is well tolerated in patients with previouslytreated MM and produces objective responses including CRs(23).

IFN-20-C2-2b MoAb

Available data indicate that progression-free survival of MMpatients is improved with IFNa, but although IFNa can havedirect cytotoxic action on plasma cells, promote both innateand adaptive immunity, and inhibit angiogenesis, its effica-ciousness as an anticancer drug has been limited due to itsshort circulating half-life and systemic toxicity (24).Rossi et al. reported the generation of the first bispecific

MoAb-IFNa, designated 20-C2-2b, which comprises twocopies of IFNa2b and a stabilized F(ab)2 of hL243 (human-

ized anti-HLA-DR; IMMU-114) site specifically linked toveltuzumab (humanized anti-CD20). In vitro, 20-C2-2binhibited eight myeloma cell lines and was more effectivethan monospecific CD20-targeted MoAb-IFNa or a mixturecomprising the parental antibodies and IFNa in all but one(HLA-DR�/CD20�) myeloma line, suggesting that 20-C2-2bshould be useful in the treatment of MM. 20-C2-2b displayedgreater cytotoxicity against KMS12-BM (CD20+/HLA-DR+

myeloma) compared with monospecific MoAb-IFNa, whichtargets only HLA-DR or CD20, indicating that all three com-ponents in 20-C2-2b could contribute to toxicity (25).

Anti-CS1 MoAb

The cell surface glycoprotein CS1 (CD2 subset 1, CRACC,SLAMF7, CD31, or 19A24), a member of the signalinglymphocyte-activating molecule–related receptor family, isselectively and constantly expressed at high levels onCD138-purified plasma cells from the MM patients (>97%),independent of the presence of metaphase cytogenetic abnor-malities or molecular subgroup. In addition, low levels ofcirculating CS1 are present in myeloma patient sera. Exceptactivated B, NK, CD8+ T cells, and mature dendritic cellswith low expression levels, it is not expressed by normal tis-sues or stem cells (26–28). Moreover, it regulates NK cellcytolytic activity via recruiting EWS-activated transcript-2and activating the PI3K/PLCg signaling pathways (29, 30).Studies propose that CS1 localizes to the uropods of polar-

ized myeloma cells suggesting a possible function for CS1 inmediating adhesion of myeloma cells to bone marrow stroma.CS1 also seems to preserve myeloma cell lines from apoptosisby lowering phosphorylation of ERK1/2, AKT, and STAT aswell as regulating pro- and anti-apoptotic pathways (31).CS1 may then contribute to MM pathogenesis by increas-

ing MM cell adhesion, clonogenic growth, and tumorigenic-ity via c-Maf-mediated interactions with BMSCs (32).The humanized anti-CS1 MoAb elotuzumab exerts antimye-

loma activity in vitro via ADCC mediated by NK cells anddoes not depend on complement-mediated cytotoxicity (33).In fact, elotuzumab caused significant ADCC against MM

cells even in the presence of BMSCs. Moreover, it provokedautologous ADCC against primary MM cells resistant totraditional or new drugs including HSP90 inhibitor and bort-ezomib and, considerably, increased HuLuc63-induced MMcell lysis when pretreated with conventional or new anti-MM therapies (34).Administration of elotuzumab causes tumor regression in

myeloma xenograft mouse models (27). Furthermore, combi-nation of elotuzumab with bortezomib significantly increasesthe in vivo therapeutic efficacy to eradicate patient-derivedmyeloma cells in a SCID-hu mouse model (34).In a phase 1, multicenter, dose-escalation study of elot-

uzumab in patients with advanced MM, patients withrelapsed/refractory MM were treated with intravenous elot-


Allegra et al. Monoclonal antibodies and multiple myeloma

uzumab. The most common AEs were cough, headache,back pain, fever, and chills. Adverse events were generallymild to moderate in severity. Nearly 26.5% had stable dis-ease (35).In other studies, most AEs attributable to elotuzumab

included mild-to-moderate infusion reactions, and the imple-mentation of more aggressive premedication regimensappeared to reduce the rate of infusion-related AEs (36, 37).Elotuzumab has been associated with different drugs,

some of which have been shown to increase its ADCC activ-ity in vitro. Pretreatment with dexamethasone or new drugssuch as bortezomib, lenalidomide, MEK inhibitors, or Aktinhibitors has been shown to significantly increase elot-uzumab-induced ADCC against MM cells (26, 34, 38).Preliminary results of clinical trials of HuLuc63 in combi-

nation with bortezomib or lenalidomide or dexamethasonewere reported at the ASH meeting 2009 (39, 40), suggestingthat elotuzumab may increase the activity of bortezomib andlenalidomide in treating MM with acceptable toxicity.Rhee et al. investigated whether the activity of elot-

uzumab could be increased by bortezomib. In vitro bortezo-mib pretreatment of myeloma targets significantly increasedelotuzumab-mediated antibody-dependent cell-mediated cyto-toxicity (ADCC), both for OPM2 myeloma cells using NKor peripheral blood mononuclear cells (PBMC) from controlsand for primary myeloma cells using autologous NK effectorcells. In an OPM2 myeloma xenograft model, elotuzumab incombination with bortezomib revealed significantly increasedin vivo antitumor activity (34).A phase 1 study evaluated elotuzumab, lenalidomide,

and dexamethasone in patients with relapsed or refractoryMM. The most frequent grades 3 to 4 toxicities were neu-tropenia (36%) and thrombocytopenia (21%). Objectiveresponses were obtained in 82% of treated patients. Thecombination of elotuzumab, lenalidomide, and low-dosedexamethasone was generally well tolerated and showedpromising response rates in patients with relapsed orrefractory MM (41).In phase 1/2 studies in relapsed/refractory MM, elot-

uzumab and bortezomib were administered. The most fre-quent grades 3 to 4 adverse events were lymphopenia andfatigue. Two elotuzumab-related serious AEs (chest pain andgastroenteritis) occurred in one patient. An objectiveresponse was observed in 48% of evaluable patients and in67% of three patients refractory to bortezomib. Median timeto progression was 9.46 months (42).

Anti-CD138 MoAb

CD138 (syndecan-1) is a heparan sulfate proteoglycan thatserves as a receptor for epidermal growth factor (EGF)ligands. Binding of EGF ligands stimulates cell growth (43).Moreover, the large extracellular domain of CD138 binds

via its heparin sulfate chains to other soluble extracellular

molecules, including the fibroblast growth factor and hepato-cyte growth factor, and to insoluble extracellular molecules,such as collagen and fibronectin. CD138 also mediates cell–cell adhesion through interactions with heparin-bindingmolecules. Studies of plasma cell differentiation show thatCD138 is a differentiation antigen and a coreceptor for MMgrowth factors (44).Immunohistochemical and flow cytometric analysis of

patient MM cells has shown that CD138 is expressed in alarge majority of cases. Within the hematopoietic compart-ment, CD138 expression is confined to normal plasma cells,with no expression on hematopoietic stem cells (HSC),while expression of CD138 on MM cells is significantlyhigher than on normal plasma cells (45).Almost all MM cells, even after exposure to multiple ther-

apies, express the antigen, making it a useful target at anystage of the disease. The extracellular components of CD138and the heparan sulfate side chains may be shed from thecell surface. Soluble syndecan-1 in serum serves as a prog-nostic indicator in MM (46).Different monoclonal antibodies (i.e., B-B4, BC/B-B4,

B-B2, DL-101, 1 D4, MI15, 1.BB.210, 2Q1484, 5F7, 104-9,281-2) specific to CD138 have been reported.Ikeda et al. showed the antitumor efficacy of three new

anti-CD138 antibody–maytansinoid conjugates, nBT062-SMCC-DM1, nBT062-SPDB-DM4, and nBT062-SPP-DM1,which vary in the linkage between the maytansinoid moietyand MoAb. The nBT062-SMCC-DM1 linkage contains athioether bond, which is not cleavable by disulfide exchange,whereas the nBT062-SPDB-DM4 and nBT062-SPP-DM1conjugates contain disulfide linkages, which can be cleavedby disulfide exchange, resulting in liberation of active may-tansinoid agent. The anti-CD138 antibody nBT062 is a mur-ine/human chimeric form of B-B4. The observed preclinicalantitumor activity of the nBT062–maytansinoid conjugatesprovides the framework for clinical development of theseagents to improve patient outcome in MM (47).Anti-CD138 immunoconjugates significantly inhibited

growth of MM cell lines and primary tumor cells from MMpatients without cytotoxicity against PBMC from healthycontrols. In MM cells, they induced G2–M cell cycle arrest,followed by apoptosis associated with cleavage of caspase-3,caspase-8, caspase-9, and poly(ADP-ribose) polymerase.Non-conjugated nBT062 completely blocked cytotoxicityinduced by nBT062–maytansinoid conjugate, confirming thatspecific binding is required for inducing cytotoxicity. More-over, nBT062–maytansinoid conjugates blocked adhesion ofMM cells to BMSC. The coculture of MM cells with BMSCprotects against dexamethasone-induced death but had noeffect on the cytotoxicity of immunoconjugates. Importantly,nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantlyinhibited MM tumor growth in vivo and prolonged hostsurvival in both the xenograft mouse models of humanMM and SCID-hu mouse model (47).



Tassone et al. first reported the potential of tumor target-ing with an anti-CD138 antibody–maytansinoid conjugateusing the murine parent of the antibody (B-B4) found inBT062. Treatment of CD138-positive cells with B-B4-DM1significantly reduced cell survival in a dose-dependent man-ner, while B-B4 antibody alone had little efficacy (48).Antitumor activity of B-B4-DM1 was also assessed in MM

xenograft studies in mice. Marked tumor regressions wereobserved upon treatment with B-B4-DM1 in subcutaneousMM models as measured by tumor volume or by fluorescentimaging of green fluorescent protein–expressing tumors. Theantitumor activity of B-B4-DM1 was proved in a SCID-humodel of human MM, where patient MM cells proliferate ina human bone chip microenvironment (49).Recently, Rousseau et al. reported preliminary biodistribu-

tion and dosimetry results obtained in refractory MMpatients in a phase 1/2 RAIT study using iodine-131-labeledanti-CD138 (B-B4) monoclonal antibody (MoAb). Fourpatients with progressive disease were enrolled after threelines of therapy. Grade 3 thrombocytopenia was documentedin two cases, and no grade 4 hematologic toxicity wasobserved. One patient experienced partial response, with60% reduction in M-spike on serum electrophoresis, andtotal alleviation of pain, lasting for 1 yr (50).

Anti-CD38 MoAb

CD38 is a 46-kDa type II transmembrane glycoprotein with ashort 20-aa N-terminal cytoplasmic tail and a long 256-aaextracellular domain. Functions ascribed to CD38 includereceptor-mediated adhesion and signaling events, as well asimportant activities that contribute to intracellular calciummobilization. Under normal conditions, CD38 is expressed atrelatively low levels on lymphoid and myeloid cells and insome tissues of non-hematopoietic origin. The relatively highexpression of CD38 on all malignant cells in MM in combina-tion with its role in cell signaling suggests CD38 as a potentialtherapeutic Ab target for the treatment of MM (51–56).Previous studies using anti-CD38 MoAb with or without

an immunotoxin (ricin) have not led to useful clinical appli-cations (57). Nevertheless, recently, a human anti-CD38IgG1 HuMax-CD38 (daratumumab) was raised after immu-nizing transgenic mice (HuMax-Mouse) possessing human,but not mouse, Ig genes. Preclinical works evidenced thatHuMax-CD38 was able in killing primary CD38+ CD138+

patient MM cells and a range of MM/lymphoid cell lines byboth ADCC and CDC (58).De Weers et al. reported the cytotoxic mechanisms of

action of daratumumab. Daratumumab induced potentADCC in CD38-expressing MM-derived cell lines as well asin patient MM cells. Daratumumab stood out from otherCD38 MoAbs in its strong ability to induce CDC in patientMM cells. Importantly, daratumumab-induced ADCC andCDC were not affected by the presence of BMSC, suggest-

ing that daratumumab can effectively kill MM tumor cells ina tumor-preserving bone marrow microenvironment. In vivo,daratumumab was highly active and interrupted xenografttumor growth at low dosing (59).Van der Veer et al. investigated the action of lenalidomide

combined with daratumumab. Daratumumab-dependent cell-mediated cytotoxicity of purified primary MM cells, as wellas of the UM-9 cell line, was significantly enhanced by lena-lidomide pretreatment of the effector cells derived fromPBMC from healthy individuals. More importantly, theydemonstrated a synergy between lenalidomide- anddaratumumab-induced antibody-dependent cell-mediatedcytotoxicity directly in the bone marrow mononuclear cells ofMM patients, indicating that lenalidomide can also potentiatethe daratumumab-dependent lysis of myeloma cells by acti-vating the autologous effector cells within the natural environ-ment of malignant cells. Finally, daratumumab-dependentcell-mediated cytotoxicity was significantly up-regulated inPBMC derived from three MM patients during lenalidomidetreatment.The lenalidomide–daratumumab combination appears to be

a highly attractive choice because these results demonstratethat lenalidomide significantly synergizes with daratumumabto improve the MM cell lysis (60).In a dose-escalation study of daratumumab in patients

with MM, the safety profile has been acceptable. In patientswith relapsed or refractory MM treated with daratumumab, amarked reduction in paraprotein and bone marrow plasmacells was observed in the higher dose cohorts. This has notpreviously been demonstrated with a single-agent monoclo-nal antibody in MM. No unexpected buildup of dar-atumumab was seen, and in patients treated with 4 mg/kgand upwards, the observed plasma concentrations were closeto those predicted. The MTD was not yet established, andthe toxicity was manageable (61).Finally, MOR202 (MorphoSysAG), a fully human anti-

CD38 IgG1 MoAb produced by a human combinatorialantibody library (HuCAL) platform, also efficiently triggersADCC against CD38+ MM cell lines and patient MM cellsin vitro as well as in vivo in a xenograft mouse model(62).

Anti-CD40 MoAb

CD40 is a transmembrane glycoprotein of the tumor necro-sis factor (TNF) receptor superfamily that is involved inB-cell activation and the formation of germinal centers (63).It is highly expressed in B-cell malignancies, such as MM,chronic lymphocytic leukemia, and NHL (64–69).CD40 activation by its ligand (CD40L) seems to partici-

pate in B-cell tumorigenesis via NFjB, ERK, p38 MAPK,and PI3-kinase signaling (70). CD40L-CD40 signaling alsoparticipates in protective tumor immunity. Thus, inhibitionof CD40L-CD40 signaling reduces tumor cell proliferation



and survival and may disrupt the protective immune stateand stimulate immune-mediated antitumor activity in MM(71, 72).Lucatumumab (HCD122, formerly CHIR-12.12) is a fully

human, recombinant IgG1 isotype monoclonal antibody thattargets CD40 and inhibits the growth and survival of B-cellmalignancies. In contrast to other anti-CD40 monoclonalantibodies that demonstrate agonist activity, lucatumumab isa full antagonist of the CD40L-binding site of CD40.Lucatumumab, in fact, binds CD40 with high affinity and

a slow off-rate and efficiently competes with CD40L toreduce B-cell differentiation. Lucatumumab also mediates anantitumor immune response by binding effector cells andinducing cell lysis via antibody-dependent cell-mediatedcytotoxicity (ADCC) in B-cell malignancies, includingCD40-positive MM cells (73–75).In an open-label, multicenter, phase 1 study lucatumumab

was evaluated in patients with relapsed/refractory MM.Common lucatumumab-related adverse events were mild-to-moderate infusion reactions. Severe adverse events wereanemia, chills, hypercalcaemia, and pyrexia (7% each). 43%of patients had stable disease, and one patient (4%) main-tained a partial response for 8 months. These findings indi-cate that single-agent lucatumumab was well tolerated up to4.5 mg/kg with modest clinical activity in relapsed/refractoryMM (76).Dacetuzumab is a different humanized anti-CD40 mono-

clonal antibody with multiple mechanisms of action. Dac-etuzumab kills tumor cells via ADCC and phagocytosis andinduction of apoptosis through direct signal transduction(77–79).Previous works have evaluated the pharmacokinetics (PK),

safety, and efficacy of dacetuzumab monotherapy in patientswith relapsed/refractory MM (80). Phase 1 data suggest it iswell tolerated with no immunogenicity (81).Treatment was generally well tolerated. The most common

adverse events were fatigue, headache, nausea, and anemia.Although transient decreases in serum and 24-h urineM-protein levels were observed for some patients, no objec-tive responses were reported.While single-agent dacetuzumab was not highly active in

this study, the observed safety profile suggested that testingdacetuzumab in combination with other MM therapies wouldbe possible. Preliminary in vitro results report that combin-ing dacetuzumab with lenalidomide is synergistic, and thiscombination may produce better response rates. Two trialsare underway in patients with relapsed MM to evaluatedacetuzumab in combination with lenalidomide or bortezo-mib (82, 83).Preclinical data have indicated that lenalidomide augments

the anti-MM efficacy of dacetuzumab, and the combinationregimen is undergoing clinical evaluation. A phase 1b studyof combination dacetuzumab plus lenalidomide and dexa-methasone in subjects with relapsed or refractory MM has

produced promising clinical response rates. The regimen wasgenerally well tolerated; fatigue, neutropenia, and thrombo-cytopenia were the most common adverse events (84).

Anti-CD56 MoAb

CD56 (neuronal cell adhesion molecule) is a membraneglycoprotein from the immunoglobulin superfamily (85),expressed on muscle cells and neurons. It appears to mediatecell adhesion, migration, invasion, and anti-apoptosis (86–88). CD 56 is also expressed on 70–90% of MM cells (89–91). Moreover, expression of CD56/neural cell adhesionmolecule correlates with the presence of lytic bone lesionsin MM and distinguishes myeloma from MGUS andlymphomas with plasmacytoid differentiation (92).Several studies have brought to the formation of anti-56

antibodies conjugated to cytotoxic moieties that combine thespecificity of antimyeloma-targeting antibodies with highlyactive antitumor compounds. One such immunoconjugatecurrently in clinical development is composed of antibodiesthat target cell surface proteins found on MM cells and arecoupled to cytotoxic maytansinoids (49).Lorvotuzumab mertansine (IMGN901) is an immunoconju-

gate composed of a humanized MoAb to CD56 (huN901)conjugated to the maytansinoid N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1), a potent antimicrotubularcytotoxic agent. HuN901-DM1 has significant in vitro and invivo anti-MM activity at low doses. Target-dependent cyto-toxicity was shown in cocultures of CD56+ and CD56� cells.Importantly, adhesion of CD56+ MM cell lines and patientMM cells to BMSCs, which is known to protect MM cellsfrom drug-induced cytotoxicity, did not protect against thespecific cytotoxicity of IMGN901. Treatment with IMGN901in a human MM tumor xenograft model in immune-compro-mised mice showed that the immunoconjugate was effectivein both a minimal and bulky disease setting (85).The phase 1 clinical study of huN901-DM1 (BB-10901)

in MM patients demonstrated an overall favorable safetyprofile (93, 94). There were no hypersensitivity reactions,and no patients developed antibodies to huN901 or DM1.Three patients achieved MR, with reductions in serum Mcomponent and urine M component. Eight patients achieveddurable stable disease, and two patients remained on treat-ment for 42 wk. Finally, additive to synergistic activity hasbeen observed in combinations of IMGN901 with lenalido-mide, bortezomib or melphalan (95, 96).

Anti-IGF-1 MoAb

The insulin-like growth factor (IGF) signaling system is com-prised of the IGF ligands (IGF-1 and IGF-2), the cell surfacereceptors that mediate the biological effects of the IGFs (IGF-1 receptor (IGF-1R), IGF-2 receptor, and the insulin receptor),as well as a family of circulating IGF-binding proteins.



IGF-1R (CD221) is expressed on MM cells in about 75%of the cases and is associated with disease outcome (97).IGF-1 produced by the microenvironment induces the activa-tion of several pathways such as phosphatidylinositol-3kinase (PI-3K)/Akt, Janus kinase (JAK)/signal transducerand activator of transduction 3 (STAT3), mitogen-activatedprotein kinase (MAPK)/extracellular signal-regulated kinase(ERK), and nuclear factor-kappa B (NF-jB).In vivo and in vitro, IGF-1 decreases drug sensitivity of

MM cells and up-regulates several anti-apoptotic proteinssuch as A1/Bfl-1, XIAP, and Bcl-2 and causes a reductionin pro-apoptotic proteins (such as caspase 3, caspase 8, cas-pase 9) and plays a role in drug resistance (dexamethasone,rapamycin) (98–103).Monoclonal antibodies targeting the IGF-1R constitute a

very attractive approach to block the IGF-1/IGF-1R interac-tion, subsequently blocking the stimulation of signalizationpathways, especially PI-3K/Akt and NF-jB (104).Tagoug et al. analyzed the effect of the combination of

the IKK2 inhibitor AS602868 (an anilinopyrimidine deriva-tive and adenosine triphosphatase competitor selected for itsinhibitory effect on IKK2 in vitro) and a monoclonal anti-body directed against IGF-1R on MM cell lines. They foundthat anti-IGF-1R potentiated the apoptotic effect ofAS602868 in LP1 and RPMI8226 MM cell lines, whichexpress high levels of IGF-1R. Anti-IGF-1R enhanced theinhibitory effect of AS602868 on NF-jB pathway signalingand potentiated the disruption of mitochondrial membranepotential caused by AS602868 (105, 106).AVE1642 is a humanized version of the murine monoclo-

nal antibody, EM164, raised against the human IGF-1R. Ithas been shown to bind specifically, with a high affinity, tohuman IGF-1R, to inhibit IGF-1 binding and receptor activa-tion, and to down-regulate the receptor by internalizationand degradation. AVE1642 has been able to delay growthand survival of some cancer cells in vitro, human tumorxenografts in nude mice, and to inhibit proliferation andsurvival of most MM cells (107, 108).Moreau et al. reported the results of a phase 1, multicen-

ter, open-label study, made to evaluate the effects ofAVE1642 alone and in combination with bortezomib, inpatients with relapsed MM. Despite the good toxicity profileof the antibody, the response rates for patients treated withAVE1642 in this study, as a single agent or in combinationwith bortezomib, were considered insufficient (109, 110).Other IGF-IR MoAbs in clinical trials in patients with

advanced solid tumors include AMG 479, IMCA12 (111,112), and R1507 (113, 114). All these antibodies vary withregard to their IgG subclass and pharmacokinetic properties,but do share some similarities. Generally, they have anadvantageous toxicity profile, and no dose-limiting toxicitieshave been described (115).The first-in-human study of figitumumab (CP-751,871)

was conducted in patients with refractory myeloma. No

dose-limiting toxicities (DLTs) were observed, althoughindividual cases of grade 3 adverse events of hyperglycemiaand anemia were seen. Of the 27 patients who received bothfigitumumab and dexamethasone, nine responded (116).Finally, dalotuzumab (MK-0646; h7C10), is a recombinant

humanized IgG1 MoAb against the IGFR1. Preliminary datafrom phase I clinical trials suggest that dalotuzumab is safe,well tolerated, and significantly inhibits tumor proliferation.Several clinical trials evaluating dalotuzumab, alone and incombination with other anticancer agents, were ongoing inpatients with MM (117).

Anti-HM1.24 (anti-317) MoAb

The HM1.24 antigen (HM1.24) is a transmembrane proteinthat has unique topology with two membrane anchordomains: an NH2-terminal transmembrane domain and a gly-cosylphosphatidylinositol attached to the COOH terminus(118). HM1.24 (CD317) was originally identified as a cellsurface protein differentially overexpressed on MM cells andlater was found to be identical to bone stromal cell antigen2 (BST-2) (119, 120).A role of HM1.24 in trafficking and signaling between the

intracellular and cell surface of MM cells was suggestedbecause it is one of the important activators of NF-jB path-way.A humanized MoAb specific to HM1.24 antigen has been

developed. Injection of the MoAb significantly reduces M-protein levels in sera and tumor cell numbers in BM andprolongs survival of myeloma-bearing mice in myelomaxenograft mouse models (121). However, its antimyelomaactivity is diminished when the mice are pretreated withanti-Fcc receptor III/II antibodies, indicating that anti-HM1.24 MoAb kills myeloma cells via ADCC and/or CDC(121–123).Single intravenous injection of AHM significantly inhib-

ited tumor growth in both orthotopic and ectopic humanMM xenograft models. A phase 1/2 clinical study showedthat a humanized anti-HM1.24 MoAb did not cause any seri-ous toxicity when administered to patients with relapsed orrefractory MM (123).Ishiguro et al. produced defucosylated AHM and evalu-

ated it by performing autologous ADCC assays against pri-mary MM cells from patients. Defucosylated AHM showedsignificant ADCC activity against three of six primary MMcells in the presence of autologous PBMC, whereas conven-tional AHM did not. The results indicate that the potency ofAHM to induce ADCC against primary MM cells was insuf-ficient, but was significantly augmented by defucosylation(124).Tai et al. investigated in vitro and in vivo anti-MM activi-

ties of XmAb5592, a humanized anti-HM1.24 MoAb withFc domain engineered to significantly enhance FccR bindingand associated immune effector functions. XmAb5592



increased ADCC several folds relative to the anti-HM1.24IgG1 analog against both MM cell lines and primary patientmyeloma cells. XmAb5592 also augmented antibody-dependent cellular phagocytosis (ADCP) by macrophages.NK cells became more activated by XmAb5592 than theIgG1 analog, evidenced by increased cell surface expressionof granzyme B-dependent CD107a and MM cell lysis, evenin the presence of BMSCs. XmAb5592 potently inhibitedtumor growth in mice bearing human MM xenografts viaFccR-dependent mechanisms and was significantly moreeffective than the IgG1 analog. Lenalidomide synergisticallyenhanced in vitro ADCC against MM cells and in vivotumor inhibition induced by XmAb5592. A single dose of20 mg/kg XmAb5592 effectively depleted both blood andbone marrow plasma cells in cynomolgus monkeys (125,126).Indeed, it is conceivable that HM1.24 may be a promising

target antigen for a cytotoxic antibody in the treatment ofMM (127).

Anti-CD48 MoAb

CD48 is a 47-kD glycophosphatidylinositol-linked glycopro-tein that is expressed on mature lymphocytes and mono-cytes, but not on non-hematopoietic tissues (128).Hosen et al. found that CD48 was highly expressed on

MM plasma cells. In 22 of 24 MM patients, CD48 wasexpressed on more than 90% of MM plasma cells at signifi-cantly higher levels than it was on normal lymphocytes andmonocytes. CD48 was only weakly expressed on someCD34+ hematopoietic stem/progenitor cells and notexpressed on erythrocytes or platelets.Administration of the anti-CD48 MoAb significantly

inhibited tumor growth in severe combined immunodeficientmice inoculated subcutaneously with MM cells. Further-more, anti-CD48 MoAb treatment inhibited growth of MMcells transplanted directly into murine bone marrow. Finally,they demonstrated that the anti-CD48 MoAb did not damagenormal CD34+ hematopoietic stem/progenitor cells (129).However, a major concern regarding CD48 as a therapeutic

target is its broad expression on normal lymphocytes andmonocytes, which may induce severe cytopenia and immuno-suppression when anti-CD48 MoAb is used as a therapeuticdrug. The potential hematologic toxicity of anti-CD48 MoAbshould therefore be very carefully tested, and anti-CD48MoAb may not be suitable for long-term maintenance ther-apy because of hematologic toxicities. For induction therapy,it could be useful for the eradication of MM plasma cells.

Anti-b2 microglobulin MoAb

b2-microglobulin (b2M) is an 11.6-kDa non-glycosylatedpolypeptide composed of 100 amino acids. It is part of theMHC class I molecule on the cell surface of nucleated cells.

Its best characterized function is to interact with and stabi-lize the tertiary structure of the MHC class I a-chain (130).High levels of serum b2M are present in hematologic

malignancies, including MM, and correlate with a poor out-come regardless of a patient’s renal function (131, 132).Anti-b2M MoAbs have been developed recently (133).

Several studies demonstrated that anti-b2M MoAbs haveremarkably strong tumoricidal activities to kill all examinedmyeloma, including tumor cell lines and primary CD138+

malignant plasma cells isolated from subjects with MM, andb2M

+/HLA-ABC+ hematologic malignant cells. Furthermore,they provoke a direct induction of tumor cell death withoutthe need for exogenous immunologic effector cells and/ormolecules, and they have capacity to kill chemotherapy-refractory myeloma. They demonstrated therapeutic efficacyin vivo in xenograft mouse models of myeloma and killmyeloma cells in the presence of BMSCs. Although themechanisms of its action in myeloma cell death need to befurther investigated, evidence has shown that anti-b2MMoAbs directly induce tumor cell apoptosis without immu-nologic effector mechanisms (133).b2M/MHC class I complex has been shown to operate as

an important signal-transducing molecule, which is involvedin responses ranging from anergy and apoptosis to cell pro-liferation and IL-2 production (134, 135). Earlier studiesshowed that cross-linking of b2M/MHC class I induces arise of intracellular free calcium concentration and activationof STAT3 and/or JNK through phosphorylation of a signal-ing motif at tyrosine 320 residue in the cytoplasmic domainof MHC class I a-chain (136, 137). It was evidenced thatbinding of anti-b2M MoAbs to myeloma cells results ininternalization and down-modulation of surface b2M/MHCclass I molecules and induction of myeloma cell apoptosis(133). Knockdown of surface b2M by specific small interfer-ence RNAs (siRNAs) significantly abrogates the MoAb-induced tumor cell apoptosis. As a result, cross-linking ofb2M/MHC class I molecules by the MoAbs activates JNKand inhibits PI3K/Akt and ERK, leading to compromisedmitochondrial integrity, cytochrome c release, and activationof the caspase-9 cascade (133). However, the mechanismsunderlying the MoAb-mediated binding to and cross-linkingof surface b2M/MHC class I molecules, and transduction ofapoptotic signals to cells needs further investigation (138).Nevertheless, IgG anti-b2M MoAbs induced apoptosis in

up to 90% of cells in a 48-h culture in all tested humanmyeloma cell lines (HMCLs) and primary myeloma cellsfrom patients. Anti-b2M MoAb-induced apoptosis in mye-loma cells was not blocked by soluble b2M, IL-6, or othermyeloma growth factors and was stronger than apoptosisobserved with chemotherapy drugs currently used to treatMM (e.g., dexamethasone) (139).Finally, Cao et al. hypothesized that IgM anti-b2M

MoAbs might have stronger apoptotic effects because oftheir pentameric structure. Compared with IgG MoAbs, IgM



anti-b2M MoAbs exhibited stronger tumoricidal activity invitro against myeloma cells and in vivo in a human-likexenografted myeloma mouse model without damaging normaltissues. IgM MoAb-induced apoptosis is dependent on thepentameric structure of the MoAbs. Disrupting pentamericIgM into monomeric IgM significantly reduced their abilityto induce cell apoptosis. Monomeric IgM MoAbs were lessefficient at recruiting MHC class I molecules into and exclu-sion of cytokine receptors from lipid rafts, and at activatingthe intrinsic apoptosis cascade (140).

Anti-CD70 MoAb

CD70 is a member of TNF family. Interaction of CD70 andits ligand CD27 modulates the expansion and differentiationof effector and memory T-cell populations (141) and pro-motes B-cell expansion and plasma cell differentiation (142).CD70 is only transiently expressed on activated B cells, Tcells, mature dendritic cells, and thymic medulla stromalcells, but it is not expressed in other normal, non-hematopoi-etic tissues (1). However, aberrant CD70 expression intumor cells has been reported in NHL, B-cell lymphocyticleukemias, Waldenstr€om macroglobulinemia, and MM (143).SGN-70, a humanized MoAb specific to CD70, has been

developed (144). SGN-70 exhibits potent antimyeloma activ-ity in vitro and significantly prolonged the survival of tumor-bearing mice in vivo. This MoAb induces Fc-mediated effectorfunctions, such as ADCC, complement fixation, and CDC.

Anti-CD74 MoAb

CD74 is an integral membrane protein that was described in1970 as an invariant chain (Ii) of the major histocompatibil-ity complex II (MHC II) in both humans and mice (145,146). It may interact with macrophage migration inhibitoryfactors that are critical mediators of the host defense and isinvolved in both acute and chronic response (147). Recentstudies have shown that CD74 is frequently expressed inMM (148). Malignant plasma cells from 80% of MMpatients and from majority of myeloma cell lines expressCD74 mRNA and protein.Moreover, CD74 protein is expressed in more than 85%

of NHL and CLL, while it shows limited expression innormal human tissues (148).Milatuzumab is a humanized anti-CD74 MoAb that

shows selective binding and rapid internalization into CD74-positive cancer cells. Milatuzumab causes growth inhibitionand induction of apoptosis in CD74-expressing MM cell lineswhen cross-linked with an anti-human immunoglobulin G sec-ondary antibody (149). Moreover, milatuzumab demonstratedpromising therapeutical activity in a CAG-SCID mouse modelofMM when used alone or in combination with doxorubicin,dexamethasone, bortezomib, or lenalidomide. Treatment ofCAG and KMS-11 MM tumors with milatuzumab, bortezomib,

and combination of both agents showed a significantly greaterefficacy of milatuzumab than that of bortezomib for treatmentof these tumors. Additionally, the combination of both agentswas even more efficacious (150, 151).In a phase I trial, milatuzumab showed no severe adverse

effects in patients with relapsed/refractory MM, and it stabi-lized the disease in some patients for up to 12 wk.A multicenter trial evaluated milatuzumab for treatment of

relapsed or refractory MM in patients who had received twoprevious standard therapies. Milatuzumab was administeredtwice weekly for 4 wk. There was a reported dose-limitingtoxicity infusion reaction and severe adverse effects (SAEs),which included a case of bacterial meningitis, fever, unex-plained hemoglobin drop, cord compression confusion,hypercalcemia, and thrombocytopenia (152).Antagonistic targeting of CD74 by milatuzumab could

remove autocrine prosurvival signals and reinstate Fas-medi-ated apoptotic signaling.

Anti-HLA-DR MoAb

Additional MoAbs are directed against a variety of furtherMM cell targets including HLA-DR by 1D09C3 (153) andHLA class I by 2D7-DB (154).NK cells play a critical role in ADCC to lyse tumor target

cells via therapeutic monoclonal antibodies, inhibitory-cellkiller immunoglobulin-like receptors (KIRs) negatively regu-late NK cell-mediated killing of HLA class I–expressingtumors, and MoAbs targeting KIR might prevent their inhib-itory signaling leading to enhanced ADCC. A new fullyhuman anti-KIR blocking MoAb, 1-7F9 (or IPH 2101),antagonizes inhibitory KIR signaling, activates NK cells,and augments NK-mediated killing of tumor cells (155,156). Importantly, 1-7F9 increases patient NK cell cytotoxic-ity against autologous MM tumor cells in vitro and appearssafe in an ongoing phase I clinical trial (157). A multicenter,open-label phase IIa clinical trial has started to evaluate IPH2101 as a single agent in patients with stable measurableMM after induction therapy. Another phase II clinical trialto assess the potential of lenalidomide combined with 1-7F9will be initiated in patients with MM (157).KIRs are receptors expressed on NK cells and a subset of

T cells and function as key regulators of NK cell activity(158). Several studies are currently underway in smolderingand first relapse MM (NCT01222286, NCT01217203,NCT00999830, NCT01248455), and safety and tolerabilityresults are expected for a phase 1 study in relapsed or refrac-tory MM (NCT00552396).Kimura et al. demonstrated that HLA class I molecules

are overexpressed in MM cells and that a recombinantsingle-chain Fv diabody against this molecule efficientlycross-links HLA class I and induces rho-mediated actinaggregation, which leads to MM cell death without effectorfunction (154, 159).



Anti-CD229 MoAb

The family of signaling lymphocytic activation molecules(SLAM) consists of nine leukocyte cell surface molecules[CD229, CD48, CD48-H1, CD84, CD150 (SLAM), CD244,BLAME, CS1, and NTB-A], which are members of theimmunoglobulin superfamily and are involved in lymphocyteactivation (160, 161).The SLAM family member CD229, which has been

shown to interact with its intracellular adapter protein Grb2in a phosphorylation-dependent manner, showed the strong-est overexpression/phosphorylation in all myeloma cell lines(162).Atanackovic et al. identified CD229 as the most strongly

over-expressed/phosphorylated immunoreceptor in myelomacell lines. Overexpression was further demonstrated in theCD138-negative population, which has been suggested torepresent myeloma precursors, as well as on primary tumorcells from myeloma patients. Accordingly, CD229 stainingof patients’ bone marrow samples enabled the identificationof myeloma cells by flow cytometry and immunohistochem-istry. Down-regulation of CD229 led to a reduced numberof viable myeloma cells and clonal myeloma colonies andincreased the antitumor activity of conventional chemothera-peutics. Targeting CD229 with a monoclonal antibodyresulted in complement- and cell-mediated lysis of myelomacells (163).

Anti-GM-2 ganglioside MoAb

GM-2 is a ganglioside expressed on MM cells. A human-ized anti-GM-2 MoAb, BIW-8962, has demonstrated in vitrokilling of MM cell lines and in vivo effectiveness in mousexenograft models, with ADCC and CDC the most prominentcytotoxic mechanisms. BIW-8962 is being evaluated asmonotherapy in a phase 1/2 study for patients with relapsed/refractory MM (164).

Anti-CD200 MoAb

CD200 is a highly conserved transmembrane glycoproteinexpressed on a wide range of cell types; however, expres-sion of the receptor for CD200 (CD200R1) is apparentlyconfined to antigen-presenting cells of myeloid lineage andcertain T-cell populations and is thought to deliver inhibitorysignals. Several studies have shown that CD200 imparts animmunoregulatory signal through CD200R, leading to thesuppression of T-cell-mediated immune responses. CD200-deficient mice have a compromised capacity to down-regu-late the activation of antigen-presenting cells (165).Moreaux et al. identified that CD200 was expressed in

malignant plasma cells in 78% of newly diagnosed MMpatients. The expression of the CD200 gene by MM cellshas been found to be a predictor of poor prognosis inpatients with MM (166).

ALXN6000 is a humanized anti-CD200 MoAb that is cur-rently being evaluated in a phase 1/2 study in patients withMM or B-cell CLL (NCT00648739), with results expectedin the near future.

Anti-CD 54 (ICAM-1) MoAb

Intercellular adhesion molecule-1 (ICAM-1) mediates adhe-sion of myeloma cells to BMSCs and thereby participates tocell adhesion–mediated drug resistance. ICAM-1 is highlyexpressed on the plasma cell surface in the majority of MMsubjects, but is also expressed on normal cell types, includingepithelial cells, endothelial cells (ECs), fibroblasts, and sev-eral types of leukocytes (167). Expression of ICAM-1 onmyeloma cells increases after chemotherapy, and high levelsof ICAM-1 predict poor response to therapy in chemo-naivepatients. Moreover, macrophages preserve myeloma cellsfrom melphalan or dexamethasone-induced apoptosis in vitro.Zheng et al. found that macrophage-mediated myeloma drugresistance was also seen with purified macrophages frommyeloma patients’ bone marrow in vitro and was confirmedin vivo using the human myeloma-SCID mouse model. Theyshowed that ICAM-1/CD18 played an important role in mac-rophage-mediated myeloma cell drug resistance, as blockingantibodies against these molecules or genetic knockdown ofICAM-1 in myeloma cells repressed macrophages’ ability toprotect myeloma cells.BI-505 is a fully human immunoglobulin G1 antibody

specific to ICAM-1 that kills myeloma cells in vitro throughinduction of ADCC and apoptosis. In addition, BI-505 inhib-its myeloma tumor growth in mice. On the basis of thesedata, a phase 1 dose-escalation study is currently recruitingrelapsed/refractory myeloma patients to evaluate efficacy andtoxicity of single-agent BI-505 (168).

Anti-Ku MoAb

The Ku heterodimer is made up of two subunits of approxi-mately 86 and 70 kDa in higher eukaryotes, which binds toDNA. Identified in 1981 from Japanese patients with sclero-derma–polymyositis overlap syndrome, Ku has now beenfound to play major roles in many cellular processes (169).First of all, Ku86 in association with Ku70 and DNA proteinkinase C plays a critical role in DNA repair especially innon-homologous end joining where the lack of functionalKu proteins in the cell typically results in genomic instabil-ity and hypersensitivity to DNA damage as well as anincreased likelihood of tumor development and immunodefi-ciency (170–173). Moreover, Ku86 has been discovered totranslocate to the cell surface of MM cells upon CD40Ltreatment and also mediate the binding of MM cells to fibro-nectin and BMSC. Most importantly, the MoAb 5E2 direc-ted against Ku86 were seen to induced apoptosis of MMcells (174–176).



Liew et al. (177) found good-affinity antibodies againstKu86, and they can be used for further studies on MM andform the basis for further development as anticancer thera-peutic agents.

MoAbs Targeting components of bone marrow

milieu

Anti-IL6 MoAb

Autocrine and paracrine IL-6 have been seen to play a cru-cial role in growth and survival of MM cells within theBM milieu (178). In BM microenvironment, IL-6 is pre-dominantly produced by BMSCs, mediating MM cellgrowth and preventing apoptotic cell death. IL-6 stimulatesat least three different signaling pathways; Ras/MEK/ERKcascade, JAK2/signal transducer and activator of transcrip-tion (STAT-3) cascade, and PI3K/Akt cascade. Importantly,IL-6 protects against apoptotic cell death induced by a vari-ety of agents. In addition, IL-6 also controls expression ofvarious other key growth in MM subjects. For example, IL-6 plays an important role in the transcriptional regulation ofmyeloid cell leukemia (Mcl)-1, an anti-apoptotic B-celllymphoma-2 family member, a critical mediator of MM cellsurvival, and tightly regulated by the proteasome (179).Therefore, down-regulation of IL-6 signaling would alsosensitize MM cells to proteasome inhibitor-mediated apop-tosis by interfering with the induction of the HSP responseand Mcl-1(180). IL-6 is also a potent osteoclast activatingfactor (OAF) for human osteoclast precursors (181–186),while clinical studies have revealed that increased serumIL-6 concentrations in patients are associated with advancedstages of MM and short survival in patients. Therefore,blocking IL-6 signaling is a potential therapeutic strategyfor cancer (187).Several clinical approaches using MoAb directed at IL-6

and IL-6R have been reported (Table 2). In the past, anti-IL-6-neutralizing MoAbs have been reported to exert notable invitro anti-MM activity. However, their in vivo and clinicaleffectiveness remains ambiguous. For example, in a phase Istudy, using a mouse–human chimeric monoclonal anti-IL6antibody, none of the MM patients achieved a response(188).However, although the clinical activity of single-agent

anti-IL-6/IL-6R in MM patients has been limited, any clini-cal studies of the anti-IL-6 monoclonal antibody CNTO 328have shown evidence of activity (189, 190).Siltuximab, formerly CNTO 328, a chimeric human–

mouse monoclonal IL-6-neutralizing antibody, has, in fact,demonstrated promising antimyeloma activity (191–193).Moreover, two recent clinical studies on CNTO 328, onein combination with dexamethasone and another with bort-ezomib, have shown evidence of encouraging activity(180).

Voorhees et al. evaluated whether CNTO 328 couldenhance the apoptotic activity of dexamethasone (dex) in pre-clinical models of myeloma. CNTO 328 potently increasedthe cytotoxicity of dex in IL-6-dependent and IL-6-indepen-dent HMCLs, including a bortezomib-resistant HMCL. Isobo-logram analysis revealed that the CNTO 328/dex combinationwas highly synergistic. Addition of bortezomib to CNTO 328/dex further enhanced the cytotoxicity of the combination.Although CNTO 328 alone induced minimal cell death, itpotentiated dex-mediated apoptosis, as evidenced by increasedactivation of caspase-8, caspase-9, and caspase-3, annexin Vstaining, and DNA fragmentation. The ability of CNTO 328to sensitize HMCLs to dex-mediated apoptosis was preservedin the presence of human BMSC (192).Hunsucker et al. reported that the combination of siltux-

imab and melphalan attenuated cell proliferation in an addi-tive to synergistic manner and enhanced apoptosis inHMCLs. This increased cell death correlated with enhancedBak activation, and siltuximab also inhibited IL-6 activationof the prosurvival PI3-K/Akt signaling pathway. Importantly,the siltuximab/melphalan combination was also effective inpatient-derived myeloma samples and partially overcamemelphalan resistance (194).The anti-interleukin-6 receptor monoclonal antibody toc-

ilizumab (TCZ) was reported to inhibit in vitro proliferationof cloned and freshly isolated myeloma cells from patientswith advanced MM (195). In addition, Nishimoto et al.(196) reported the first 2 cases of refractory MM treatedwith TCZ, noting that it improved fever and systemic edemaand also stabilized the monoclonal protein levels.Matsuyama et al. reported a patient with rheumatoid

arthritis (RA) and smoldering MM, in whom TCZ improvedRA symptoms and also stabilized the serum levels of mono-clonal IgA (197).Tocilizumab treatment is generally well tolerated and safe.

It is now evaluated in open-label phase I (USA) and II(France) trials to assess its safety and efficacy as monothera-py in MM patients who are not candidates for, or who haverelapsed after SCT (197).In addition, NRI, another receptor inhibitor of IL-6 geneti-

cally engineered from tocilizumab, is under preclinical evalua-

Table 2 Monoclonal antibodies targeting components of bone

marrow milieu

Target Name

IL-6 Siltuximab (CNTO328)

Tocilizumab

NRI

Elsilimomab (B-E8)

Azintrel (OP-RR003-1)

SANT-7

VEGF Bevacizumab

EGFR Cetuximab

FGFR-3 MFGR1877A



tion (198). An adenovirus vector encoding NRI was adminis-tered to mice intraperitoneally (i.p.) and monitored for theserum NRI level and growth reduction property on the xeno-grafted IL-6-dependent MM cell line S6B45.Elsilimomab is a murine monoclonal antibody, also known

as B-E8, which has been studied in hematologic malignancies(199–201). Initial studies of BE-8 demonstrated a transienttumor cytostasis and reduction in toxicities from IL-6 (202).The potential of combination therapy, including BE-8

(250 mg), Dex (49 mg/d), and high-dose melphalan[220 mg/m2 (HDM220)], followed by autologous SCT wasdemonstrated. 81.3% of patients exhibited a response, with acomplete response seen in 37.5% of patients without anytoxic or allergic reactions.A high-affinity fully human version of BE-8, OP-R003-1

(or 1339, Azintrel), was studied. It enhanced cytotoxicityinduced by dexamethasone, as well as bortezomib, lenalido-mide, and perifosine, in a synergistic fashion. Importantly,Azintrel also blocked bone turnover in SCID-hu mousemodel of MM, providing an additional rationale for its usein MM (203).Finally, IL-6R antagonist SANT-7, in combination with

Dex and all-trans retinoic acid (ATRA) or zoledronic acid,strongly inhibited growth and induced apoptosis in MM cells(204–206).

Anti-VEGF MoAb

One key compartment of the MM microenvironment isthe vascular niche. The role of angiogenesis in hematolog-ic malignancies is now well established. VEGF within theMM BM microenvironment induces growth, survival aswell as migration of MM cells in an autocrine manner viaVEGFR-1 and triggers angiogenesis via VEGF-2 in ECs.Recent works suggest the existence of MM-specific ECs(MMECs), which produce growth and invasive factors forplasma cells, including VEGF, FGF-2, MMP-2, as well asMMP-9. Compared with healthy human umbilical vein EC(HUVEC), MMECs secrete higher amounts of the CXCchemokines (e.g., IL8, SDF1-a, MCP-1), which act in aparacrine manner to mediate plasma cell proliferation andchemotaxis. Moreover, MM cells and BBMSC prolongsurvival of ECs both by increased secretion of EC survivalfactors, such as VEGF, and by decreased secretion ofanti-angiogenic factors (183, 207–210). Research on angio-genesis has led to the clinical approval of several anti-angiogenic agents in MM.Bevacizumab blocks VEGF and VEGF’s binding to its

receptor on the vascular endothelium (211). Anti-VEGF Abswere active alone and in combination with radiation in earlierpreclinical studies (212). It is currently being studied clinicallyin many diseases as systemic amyloidosis and MM (213).NCI’s Cancer Therapy Evaluation Program is sponsoring aphase II study of bevacizumab plus thalidomide in MM (214).

Attar-Schneider et al. explored the efficacy of anti-VEGFtreatment with bevacizumab directly on MM cells. Theyshowed that blocking VEGF is detrimental to the MM cellsand provokes cytostasis. This was evidenced in MM celllines, as well as in primary BM samples (BM MM). Utiliz-ing a constitutively Akt-expressing MM model, they showedthat the effect of bevacizumab on viability and eIF4E statusis Akt dependent (215).Somlo et al. tested the efficacy of bevacizumab alone and

in combination with thalidomide in MM patients with aphase II prospective randomized/stratified trial. Toxicitieswere mild. Among those who received bevacizumab alone,one patient – with the greatest expression of VEGF on MMcells – achieved stable disease for 238 d, but the medianEFS for the cohort was only 49 d (216).

Anti-EGFR MoAb

The epidermal growth factor receptor (EGFR) is a member ofthe ErbB family of transmembrane tyrosine kinase receptorsand contributes to malignant cell survival and proliferation(217).Upon ligand binding, the EGFR dimerizes in hetero- or

homodimers, which results in the activation of an intrinsictyrosine kinase and the initiation of activating signaling cas-cades: the Ras and MAPK pathways, and the PI-3K and pro-tein kinase B (Akt) pathways. In addition to activating thepathways that stimulate cell proliferation and cell survival,recent evidence shows that the EGFR might directly act as atranscription factor upon activation (218).Cetuximab is a chimeric human–murine monoclonal anti-

body that binds competitively and with high affinity to theEGFR. Binding of the antibody to the EGFR prevents stimu-lation of the receptor by endogenous ligands and results ininhibition of cell proliferation and angiogenesis andenhanced apoptosis. Binding of cetuximab to the receptoralso results in internalization of the antibody–receptor com-plex, which leads to an overall down-regulation of EGFRexpression (219).Cetuximab is approved for the treatment of metastatic colo-

rectal cancer and relapsed/metastatic head-and-neck-cancerand other epithelial malignancies (220, 221). Recent data indi-cate that the EGFR is expressed on the malignant plasma cellsof MM and on cells of the MM microenvironment. Moreover,MM cells coexpress the EGFR ligands amphiregulin and hep-arin-binding EGF-like growth factor (HBEGF), and inhibitionof EGFR ligand signaling induces MM cell apoptosis (222,223).B€oll et al. showed an activity of cetuximab as single agent

in a patient with relapsed MM. Cetuximab was administeredintravenously. After the initiation of cetuximab treatment,they measured disease stabilization as measured by IgG andserum-free kappa light chains in serum and urine. After atotal of 44 wk, they stopped cetuximab treatment, and they



measured an increase in both IgG and kappa light chains inserum and urine (224).

Anti-FGFR3 MoAb

At the cytogenetic level, the MM genome is recognized asbeing complex. The study of chromosomal translocationsgenerated by aberrant class switch recombination shows thatseveral oncogenes, including fibroblast growth factor recep-tor 3 (FGFR3), are placed under the control of the strongenhancers of the heavy chain Ig (IGH) loci, leading to theirderegulation (225).Overexpression of FGFR3 has been implicated in the

specific changes in gene expression observed in plasma cellsas a result of the t(4;14) translocation. Microarray analysis ofpatient samples with the t(4;14) translocation identified a spe-cific genetic signature characterized by perturbations in theexpression of several other genes. A small proportion ofpatients with the t(4;14) translocation also have FGFR3-acti-vating mutations, including A1157G, A1987G, A761G, andG1138A; however, overall these mutations are rare, with afrequency of approximately 5% among t(4;14) patients (226).As a part of the translational effort, new drugs that inhibit

oncogenic proteins overexpressed in defined molecular sub-groups of the disease, such as FGFR3 and MMSET in t(4;14) MM, are currently being developed (227).MFGR1877A is a human IgG1 monoclonal antibody

that binds to FGFR3 and is being investigated as a poten-tial therapy for relapsed/refractory FGFR3+ MM. Kamathet al. characterized the PK of MFGR1877A in mouse, rat,and monkey. PK of MFGR1877A was determined inathymic nude mice, Sprague–Dawley rats, and cynomolgusmonkeys after administration of single intravenous doses.The antitumor efficacy in mice bearing human tumorxenografts was used in conjunction with inhibitory activityin cell proliferation assays. Doses ranging from 2 to 3 mg/kg weekly to 6–10 mg/kg every 4 wk were predicted toachieve the target exposure in � 90% of MM patients(228).

MoAbs Targeting Tumor–BMSC Interaction

MM cells are highly dependent on the BM microenviron-ment for growth and survival through interactions particu-larly with BMSCs and osteoclasts, which secrete importantMM growth factors. Thus, MoAbs designed to blockthe binding of MM cell growth and survival factors to theircognate receptors have been under intensive development(Table 3).Moreover, in addition to therapy directed at MM cells and

tumor-promoting interactions, some efforts have beendevoted to MoAb therapy directed against the developmentof complications; to date, these efforts have been restrictedto the suppression of myeloma-related bone disease.

Anti-RANKL MoAb

Denosumab is an investigational fully human MoAb withhigh affinity and specificity for RANKL that mimics thenatural bone-protecting actions of OPG (229). Although de-nosumab was recently approved to treat osteoporosis andprevent the skeletal-related events in patients with bonemetastases from solid tumors (230, 231) in the United Statesand Europe, it is still undergoing phase III clinical trials ofits efficacy in treating MM-induced bone disease.A phase 1 clinical trial in patients with MM or breast can-

cer with bone metastases showed that following a single s.c.dose of denosumab, levels of urinary and serum N-telopep-tide decreased within 1 d, and this reduction lasted through84 d at the higher denosumab doses (232).The most commonly reported adverse events after denosu-

mab administration in patients with MM were fatigue, ane-mia, upper respiratory tract infection, and headache (233). Inaddition, a case of ONJ in a patient who had received deno-sumab was reported (234).Larger trials are ongoing to investigate the effect of deno-

sumab for the treatment of cancer-induced bone disease(235), and a recent in silico investigation supports the ideathat denosumab represents a convenient alternative topamidronate in the treatment of MM-induced bone disease(236).

Anti-Dickkopf-1 MoAb

Dickkopf-1 (DKK1), a soluble inhibitor of Wnt/b–cateninsignaling required for embryonic head development, regu-lates Wnt signaling by binding to the Wnt coreceptor lipo-protein-related protein-5 (LRP5)/Arrow. LRP5 mutationscausing high bone mass syndromes disrupt DKK1-mediatedregulation of LRP5. Forced overexpression of Dkk1 inosteoblasts causes osteopenia, disruption of the HSC niche,and defects in HSC function. Dkk1 also inhibits fracturerepair. Studies suggest that DKK1 activation in osteoblastsis the underlying cause of glucocorticoid- and estrogendeficiency–mediated osteoporosis and at least partiallyunderlies the teratogenic effects of thalidomide on limbdevelopment.

Table 3 Monoclonal antibodies targeting tumor–bone marrow stromal

cell interaction

Target Name

RANKL Denosumab

Dickkopf Anti-DKK1

BrlQ880

Activin RAP-011

ACE-011

BAFF Atacicept

SG1



DKK1 has been implicated in the osteolytic phenotypes ofMM. Preclinical studies have shown that neutralizingDKK1/Dkk1 and/or enhancing Wnt/b–catenin signaling mayprove effective in treating bone pathologies (237).The effect of anti-DKK1 MoAb on bone metabolism and

tumor growth in a SCID-rab system has been evaluated. Theimplants of control animals showed signs of MM-inducedresorption, whereas mice treated with anti-DKK1 antibodiesblunted resorption and improved the bone mineral density ofthe implants. Histologic examination revealed that myeloma-tous bones of anti-DKK1-treated mice had increased num-bers of osteocalcin-expressing osteoblasts and reducednumber of multinucleated TRAP-expressing osteoclasts. Thebone anabolic effect of anti-DKK1 was associated withreduced MM burden (238).A different anti-DKK1 agent is BHQ880 (239, 240).

Although BHQ880 had no direct effect on MM cell growth,BHQ880 increased osteoblast differentiation, neutralized thenegative effect of MM cells on osteoblastogenesis, andreduced IL-6 secretion. Furthermore, in a SCID-hu murinemodel of human MM, BHQ880 treatment led to a significantincrease in osteoblast number, serum human osteocalcinlevel, and trabecular bone. A preliminary result from a phaseI/II trial in MM where BHQ880 was given was well toler-ated when given in combination with zoledronic acid.Finally, Dickkopf-1 (DKK1), broadly expressed in mye-

loma cells but highly restricted in normal tissues, togetherwith its functional roles as an osteoblast formation inhibitor,may be an ideal target for immunotherapy in myeloma.Quian et al. examined whether DKK1 can be used as a

tumor vaccine to elicit DKK1-specific immunity that cancontrol myeloma growth or even eradicate established mye-loma in vivo. They used DKK1-DNA vaccine in the murineMOPC-21 myeloma model, and the results showed thatactive vaccination using the DKK1 vaccine not only wasable to protect mice from developing myeloma, but also wastherapeutic against established myeloma. Mechanistic studiesrevealed that DKK1 vaccine elicited a strong DKK1- andtumor-specific CD4+ and CD8+ immune responses, andtreatment with B7H1 or OX40 Abs significantly reduced thenumbers of IL-10-expressing and Foxp3+ regulatory T cellsin vaccinated mice (241).

Anti-activin A MoAb

Activin A is a TGF-b superfamily member most commonlyassociated with embryogenesis and gonadal hormone signal-ing (242). In addition, activin A is involved in bone remod-eling with growth stimulatory effects on osteoclasts (Ocs)(243). Activin A inhibits osteoblasts (OB) differentiation bystimulating SMAD2 activity and inhibiting distal-lesshomeobox (DLX)-5 expression. More importantly, inhibitionof activin A signaling rescued MM-induced OB impairmentin vitro and in vivo while reducing MM burden in a human-

ized myeloma model. Malignant plasma cells disrupt thenormal regulatory pathway of bone homeostasis by inducingBMSC secretion of activin A via JNK pathway (244).Activin can be targeted by a chimeric antibody RAP-011,

derived from the fusion of the extracellular domain of acti-vin receptor IIA and the constant domain of the murineIgG2a. ACE-011, a novel bone anabolic agent currently in aphase 2 clinical trial in MM, is a protein therapeutic basedon the activin receptor IIA. In MM, an ongoing multicenterphase 2 trial is conducted in patients who are treated withmelphalan, prednisone, and thalidomide. Preliminary resultsshow clinical significant increases in biomarkers of bone for-mation, improvement in skeletal metastases, and decreases inbone pain as well as antitumor activity. Moreover, ACE-011has potential as a novel therapy for chemotherapy-inducedanemia and may be an effective alternative to erythropoietin(EPO)-based treatments (245).

Anti-BAFF MoAb-

B-cell-activating factor (BAFF) is a TNF superfamily mem-ber and produced in the bone marrow microenvironment bymonocytes, osteoclasts, and neutrophils. Recently, BAFFwas recognized as new survival factors for MM (246). Inaddition to BMSCs, osteoclasts produce these factors tosupport MM cells in the BM microenvironment (247).Binding of BAFF to the three different TNF-R-related

receptors, TACI, BCMA, and BAFF-R, triggers activation ofNF-jB, PI3K, and MAPK pathways, resulting in survivaland dexamethasone resistance of myeloma cells (248, 249).BAFF also enhances adhesion of myeloma cells to BMSC

(250). In a mouse model, an anti-BAFF-neutralizing anti-body had antimyeloma activity and inhibited osteoclastogen-esis.Atacicept acts as a decoy receptor by binding to and neutral-

izing soluble BAFF and APRIL and preventing these ligandsfrom binding to their cognate receptors on B-cell tumors,thereby enhancing cytotoxicity. An open-label, dose-escalation phase I/II study enrolled patients with refractory orrelapsed MM or active, progressive Waldenstr€om macroglob-ulinemia (251, 252). Atacicept was well tolerated and showedclinical and biological activity consistent with its mechanismof action. TACI was expressed heterogeneously among patientMM cells, which may explain promising results for the treat-ment of TACIhigh MM cells in a trial for atacicept (253).On the basis of these results, a phase 1 study is currently

enrolling relapsed/refractory myeloma patients to evaluatethe combination of the neutralizing anti-BAFF antibody,LY2127399, combined with bortezomib.Finally, B-cell maturation antigen (BCMA) is expressed

on normal and malignant plasma cells and represents apotential target for therapeutic intervention. BCMA binds totwo ligands that promote tumor cell survival, a proliferationinducing ligand (APRIL) and B-cell activating factor. To



selectively target BCMA for plasma cell malignancies, Ryanet al. developed antibodies with ligand-blocking activity thatcould promote cytotoxicity of MM cell lines as naked anti-bodies or as antibody–drug conjugates. They showed thatSG1, an inhibitory BCMA antibody, blocks APRIL-dependent activation of NF-jB in a dose-dependent mannerin vitro. Cytotoxicity of SG1 was assessed as a naked anti-body after chimerization with and without Fc mutations thatenhance FcgammaRIIIA binding. The Fc mutationsincreased the antibody-dependent cell-mediated cytotoxicitypotency of BCMA antibodies against MM lines by approxi-mately 100-fold with a � 2-fold increase in maximal lysis.As an alternative therapeutic strategy, anti-BCMA antibodieswere endowed with direct cytotoxic activity by conjugationto the cytotoxic drug, monomethyl auristatin F. The mostpotent BCMA antibody–drug conjugate displayed IC(50)values of � 130 pmol/L for three different MM lines (254).

Other potential targets

Anti-TRAIL-R1 R2 MoAb

The TNF family members Fas ligand and TRAIL induceapoptosis upon binding to the death receptors Fas, andTRAIL receptor 1(TRAIL-R1; DR4), or TRAIL receptor 2(TRAIL-R2; DR5), respectively. TRAIL also binds to thedecoy receptors DcR1 and DcR2 and to the soluble decoyreceptor osteoprotegerin (OPG), which is produced by osteo-blasts and stromal MoAbs activating death receptors (255–258) (Table 4).TRAIL-R1 and TRAIL-R2 antimyeloma activity was

observed for the fully human agonistic antibody directedagainst TRAIL-R1 (mapatumumab, HGS-ETR1) and to alesser extent for the anti-TRAIL-R2 antibody (lexatumumab,HGS-ETR2). The two human agonistic MoAbs killed 68%and 45% of MM cell lines, respectively. Only 18% of MMcell lines are resistant to either antibody (259).Importantly, the antimyeloma action of anti-DR4 and anti-

DR5 antibodies was not affected by the presence of cells ofthe bone marrow microenvironment, whereas these cellsprotected myeloma cells against TRAIL-induced apoptosis(260). This protective effect was at least partly mediated byOPG.Bortezomib treatment is associated with the up-regulation

of TRAIL and its receptors in B-CLL and lymphoma cells(261). Furthermore, blockage of TRAIL-R1 and TRAIL-R2expression using RNA interference, which prevents TRAILapoptotic signaling, inhibited proteasome inhibitor-inducedapoptosis.Based on enhanced cytotoxicity when combining map-

atumumab with bortezomib in preclinical experiments (262),a randomized phase II study was recently started comparingTRM-1 plus bortezomib versus bortezomib alone in patientswith relapsed or refractory MM (263).

Indeed, combined bortezomib plus mapatumumab treat-ment resulted in enhanced myeloma cell killing when com-pared to either agent alone, in both wild-type andbortezomib-resistant cells. In contrast to the preclinical data,a randomized phase 2 study in relapsed/refractory myelomashowed that addition of mapatumumab to bortezomib treat-ment did not increase response rate or progression-free sur-vival compared with bortezomib alone (264).

Anti-PD-L1 MoAb

PD-L1 (also known as B7-H1 or CD 274) is a B7 familymember and is the ligand for PD-1 (programmed death-1),a member of the B28 family. PD-L1 interacts with PD-1and an unknown receptor on T cells and can inhibit T-cellactivation and cytotoxic T-lymphocytes-mediated lysis(265).PD-L1 overexpression appears as a possible mechanism

for tumors to avoid the host’ immune response, and a wayof improving antitumor activity of NK cells against mye-loma cells is by modulating the PD-1/PD-L1 axis, whichdown-regulates the immune response. PD-1 is not present onNK cells from healthy donors, but can be up-regulated byexogenous IL-2. In contrast, PD-1 is present on NK cellsfrom patients, and interaction with PD-L1 on myeloma cellsinhibits NK cell function. PD-L1 is expressed on plasmacells from the majority of myeloma patients, but only rarelyon plasma cells from MGUS patients and not on normalplasma cells (266). CT-011 is a humanized anti-PD-1MoAb, which enhanced NK cell function against myelomatumor cells through increased NK cell trafficking, improvedimmune complex formation between myeloma cells and NKcells, and increased NK cell cytotoxicity.Lenalidomide not only activates NK cells, but also down-

regulates PD-L1 expression on myeloma cells and synergizedwith CT-011 in the activation of NK cells and subsequentkilling of myeloma cells. Apart from effects on NK cells,blockade of the PD-1/PD-L1 interaction with anti-PD-L1MoAbs enhanced myeloma-specific T-cell immunity in vitroand in vivo (267).Furthermore, CT-011 also enhanced T-cell responses to

autologous dendritic cell/myeloma fusion vaccines (268).This may be explained by augmented expression of PD-1 onT cells from myeloma patients when compared to healthycontrols.

Table 4 Other potential targets of monoclonal antibodies

Target Name

TRAIL-R1 Mapatuzumab

TRAIL-R2 Lexatuzumab

PD-L1 CD-011

VLA-4 Natalizumab



On the basis of these data, a phase 1 clinical trial wasstarted to study the action of CT-011 in patients withadvanced hematologic malignancies including myelomapatients. CT-011 was demonstrated to be well tolerated withevidence of antitumor activity. Treatment with CT-011 wasaccompanied with an elevated percentage of peripheral bloodCD4-positive T cells.The combination of CT-011 with an IMiD may further

enhance host antitumor immunity and warrants further inves-tigation. Alternatively, the immune response to myelomamay be enhanced by anti-PD-L1 antibodies such as MDX-1105.

Anti-VLA-4 MoAb

BMSCs as well as extra cellular matrix (ECM) proteinslaminin, microfibrillar collagen type IV, and fibronectin arestrong adhesive components for MM cells via interaction ofa variety of integrins including integrin-a4 (also CD49d, asubunit of CD49d/CD29 or very-late antigen-4; VLA-4)(269).A marked increase in BM angiogenesis in MM was found

to correlate with VLA-4 expression in plasma cells isolatedfrom patients with active MM. The ability of bortezomib toovercome cell adhesion–mediated drug resistance (CAM-DR) (vincristine and dexamethasone) is, at least in part, dueto down-regulation of VLA-4 expression (270).Directly targeting VLA-4 and its subunit integrin-a4 in

particular is therefore of high therapeutic interest in MM.Previous studies using a murine anti-integrin-a4 antibodyreduced MM growth, IgG2b production, and associatedosteoclastic osteolysis in 5TGM1 murine MM cells both invitro and in vivo (271).Podar et al. evaluated the therapeutic potential of the new-

in-class-molecule selective-adhesion molecule (SAM) inhibi-tor natalizumab, a recombinant humanized IgG4 monoclonalantibody, which binds integrin-a4, iMM (272).Natalizumab, but not a control antibody, inhibited adhe-

sion of MM cells to non-cellular and cellular components ofthe microenvironment as well as disrupted the binding ofalready adherent MM cells. Consequently, natalizumabblocked both the proliferative effect of MM–BMSC interac-tion on tumor cells and vascular endothelial growth factor(VEGF)-induced angiogenesis in the BM milieu. Moreover,natalizumab also blocked VEGF- and IGF-1-inducedsignaling sequelae triggering MM cell migration. Moreover,natalizumab inhibited tumor growth, VEGF secretion, andangiogenesis in a human severe combined immunodeficiencymurine model of human MM in the human BM microenvi-ronment.Importantly, natalizumab not only blocked tumor

cell adhesion, but also chemo-sensitized MM cells to bort-ezomib, in an in vitro therapeutically representative humanMM–stroma cell coculture system model (273).

Anti-CD11C1 (antikininogen) MoAb

Sainz et al. used two cell lines (B38 and C11C1) derivedfrom P3X63Ag8 myeloma cells. The new cell lines wereimplanted subcutaneously in the strain of origin (Balb/cmice) and used as a model to monitor the effects of C11C1MoAb to kininogen (HK). They assessed their behavior byintraperitoneal and subcutaneous implantation, by implantingthem together and by treating B38-MM with purified MoAbC11C1. They found that MoAb C11C1 inhibits its owntumor growth in vivo and slows down B38-MM growth rateboth when MM is implanted together and when MoAbC11C1 is injected intraperitoneally. MAb C11C1-treatedMM showed decreased MVD and HK binding in vivo with-out FGF-2, B1R, or B2R expression changes (274).

Polyclonal antibody (rATG)

Polyclonal antibody preparations may have several advanta-ges over monoclonal therapeutic agents, including the abilityto target multiple surface proteins and simultaneously triggerseveral parallel or additive pathways for cell death. This maybe a distinct advantage when attempting to eradicate mye-loma cells, which emerge from a common less differentiatedprecursor (275, 276), and may be responsive to coordinateactivation of several cell death pathways (277, 278).Zand et al. measured complement-independent cell death

measured after addition of polyclonal rabbit antithymocyteglobulin (rATG) to cultures of myeloma cell lines or primaryCD138+ isolates from patient bone marrow aspirates. rATGinduced significant levels of apoptosis in myeloma cells asassayed by caspase induction, annexin V binding, subdiploidDNA fragmentation, plasma membrane permeability, andloss of mitochondrial membrane potential. Three pathwaysof cell death were identified involving caspase activation,cathepsin D, and the genistein sensitive tyrosine kinase path-way. Fab′2 fragments of rATG had reduced pro-apoptoticactivity, which was restored by co-incubation with Fc frag-ments and anti-CD32 or anti-CD64 antibodies (279).

Future perspectives for monoclonal antibodies

in multiple myeloma: limits and challenges

Therapeutic monoclonal antibodies have revolutionized treat-ment options for many cancers; however, MoAbs targetingmyeloma cells have not yet been included as part of stan-dard myeloma therapy. Despite several efforts, the benefit ofMoAb-based therapy directed at different targets in MMremains incompletely articulated. MAbs, when employed asmonotherapy in MM, have generally not produced impres-sive levels of response with respect to either response ratesor extent of response in individual patients. However, pre-clinical results in MM cell lines and murine explant modelsand preliminary clinical results in patients with relapsed/refractory MM suggest that MAbs are likely to act synergis-



tically with traditional therapies (dexamethasone), immunemodulators (thalidomide, lenalidomide), and other noveltherapies (such as the first-in-class proteasome inhibitor bort-ezomib); in addition, MAbs have shown the ability to over-come resistance to these therapies (280).Moreover, along with efforts to develop functional anti-

bodies that could provide benefit to MM patients, substantialefforts are underway to develop therapies using antibodiesconjugated to potent cytotoxic agents. A variety of highlycytotoxic compounds are being evaluated for antibody-baseddelivery, including calicheamicin, doxorubicin, taxanes,maytansinoids, dolastatins, and CC-1065 analogs (281, 282).The near future will see a novel interest in developing

novel targets for antibody-based therapies for MM. BMangiogenesis has an important role in the initiation and pro-gression of MM. Berardi et al. looked at novel mechanismsof vessel formation in patients with MM through a compara-tive proteomic analysis between BM ECs of patients withactive MM (MMECs) and ECs of patients with monoclonalgammopathy of undetermined significance (MGECs) and ofsubjects with benign anemia (normal ECs). Four proteinswere found overexpressed in MMECs: filamin A, vimentin,a-crystallin B, and 14-3-3f/d protein. Berardi et al. investi-gated the differences in MMEC versus MGEC proteome toidentify new targets for MM anti-angiogenic management.They found that FLNA, VIM, CRYAB, and YWHAZ areconstantly overexpressed in MMECs and enhanced byVEGF, FGF2, HGF, and MM plasma cell CM. These pro-teins are critically involved in MMEC overangiogenic phe-notype, and indeed, their silencing is anti-angiogenic (283).However, one of the major problems with MoAb therapy is

its immunogenicity, specifically defined as human anti-mouseantibody (HAMA). To overcome this limitation, murinemolecules are engineered to remove immunogenic murinecontent, which has been initially achieved by generation ofmouse–human antibodies. Chimeric antibodies are composedof murine variable regions fused with human constant regions.Although with reduced immunogenicity and prolonged half-life, chimeric MoAbs still contain a significant proportion(approximately 35%) of antigenic mouse determinants, sug-gesting a possibility to generate HAMA.Finally, although the ability to create essentially human

antibody structures has reduced the likelihood of host-protective immune responses that otherwise limit the utilityof therapy, majority of MM patients are immunosuppressive.The immediate goal would be testing next generations ofgenetically Fc-engineered MoAbs that not only bind to targetMM antigens with high affinity but also have superior inter-action with host immune effectors.

Conclusions

Results of experimental agents targeting a number of potentialcandidate molecules expressed on the surface of MM tumor

cells have been reported as have those of MoAbs targetingother proteins involved in the MM immunologic microenvi-ronment. The introduction of novel antimyeloma agents willresult in a more individualized targeted therapy (284).It remains to be defined how MAb therapy can most

productively be incorporated into the current therapeutic para-digms that have achieved significant survival gains in patientswith MM. This will require careful consideration of the opti-mal sequence of therapies and their clinical placement aseither short-term induction therapy, frontline treatment inpatients ineligible for ASCT, or long-term maintenance ther-apy (285).

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Monoclonal antibodies: potential new therapeutic treatment against multiple myeloma

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