General considerations on monoclonal antibodies and multiple myeloma
Multiple myeloma (MM) is a progressive and fatal disease characterized by the malignant proliferation of plasma cells in the bone marrow and overproduction of monoclonal immunoglobulin or light-chain proteins. MM therapy remains challenging. Despite recent therapies, such as bortezomib, thalidomide, and lenalidomide, treatment is limited and palliative, and MM remains an incurable disease associated with a median survival of 4 yr .
Patients refractory to both bortezomib and either lenalidomide or thalidomide were found to have a median overall survival of 9 months . Then, the outcome of patients with MM treated with cytotoxic therapy has not been adequate, and new treatments are needed for relapsed or refractory MM.
A new anticancer strategy is the use of monoclonal antibodies (MoAb) that operate through completely different mechanisms of action. MoAbs directed against MM-associated markers should be taken into account in clinical practice, because they could represent the best available combination of tumor cytotoxicity, environmental signal privation, and immune system redirection .
Ideally, targets for therapeutic MoAbs should be specifically expressed on cancerous cells but not on normal cells. In fact, monoclonal antibodies are an interesting thera-peutic option in MM because they are specific to a tumor-associated target and have been successfully employed in the treatment of patients with other hematologic diseases .
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 antigen targets, which can be expressed either on myeloma cells or on components of the bone marrow microenvironment [bone marrow stromal cells (BMSCs) or signaling molecules] [3, 5].
Therapeutic MoAbs use one or more mechanisms to reduce tumor burden in patients. They could directly induce apoptosis or growth arrest upon binding to cell surface antigen on tumor cells. Rituximab and mapatumumab could cause growth inhibition or apoptosis signaling to block tumor cell growth and survival. Such mechanism of action was employed by MoAbs conjugated with toxins, that is, maytansinoids for anti-CD56 and anti-CD138, thus directly target and eliminate tumor cells.
Most of the approved therapeutic MoAbs belong to IgG1 subclass, which has a long half-life, and activate immune effector functions. After the binding of MoAbs to a specific target on a tumor cells, antibody-dependent cellular cytotoxicity (ADCC) is triggered by interactions between the Fc region of an antibody bound to a tumor cell and Fc receptors, particularly FcRI and FcRIII, on immune effector cells such as natural killer (NK) cells, macrophages, and neutrophils. MoAb-coated tumor cells are phagocytosed by macrophages or undergo cytolysis by NK cells. In the case of complement-dependent cytotoxicity (CDC), recruitment of C1q by IgG bound to the tumor cell surface is the first step. This triggers a proteolytic cascade that induces production of the effector molecule, C3b, and then formation of a membrane attack complex that kills the target cell by disrupting its cell membrane.
MoAbs have been additionally designed to functionally block both autocrine- and paracrine-secreted cytokines and growth factors as well as molecules mediating MM–stromal cell interaction.
Although expression of MoAb targets is not always confined to the myeloma cells, toxicity of the MoAbs is relatively mild. On the other hand, minimizing side effects of chemotherapy can also be achieved using myeloma-specific antibody conjugates to specifically deliver cytotoxic drugs to the tumor cells. This method will consent the administration of lower doses of drugs and may therefore reduce toxicity of antimyeloma 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 between MM cells and BMSCs .
These potential targets include mediators of adhesion, signaling molecules, cell surface receptors, and plasma cell growth factors.
MoAbs Targeting tumor cells
Several MoAbs directed against MM cell surface are being investigated as potential targets in MM patients (Table 1).
Table 1. Monoclonal antibodies (MoAbs) targeting tumor cells
|β2 m||IgG anti-β2 m|
|IgM anti-β2 m|
Several works have shown that MM includes clonotypic B lineage cells at stages earlier than the compartment of malignant plasma cells in the bone marrow , and the circulating component of the MM clone includes at least two distinct CD19+ 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 a 5-month period during and after treatment. Overall, all three types of circulating B lineage cells persist despite treatment with rituximab. The inability of rituximab to prolong survival in MM may result from this failure to deplete CD20+ B and plasma cells in MM .
Indeed, previous studies demonstrated only minimal activity of anti-CD20 rituximab and antibodies against plasma cell-specific CD38 antibodies in MM [11-13]. However, although the CD-2 subgroup of myeloma frequently overexpresses CD20 and few studies trying to prove otherwise , clinical studies of rituximab treatment in MM have for the most part been discouraging, with few patients achieving only minimal responses . Results from a clinical phase 2 trial in relapsed MM showed that rituximab treatment yielded significant reduction in circulating B cells but had no beneficial clinical effect .
Moreover, rituximab was tested for maintenance therapy in MM following autologous hematopoietic stem cell transplantation (SCT) . The use of rituximab in this sort of patients was associated with an unexpectedly high rate of early relapse. The authors then hypothesized a possible role for rituximab in provoking an additional reduction in the normal, residual B-cell activity.
MM is generally not considered as a disease adequate for anti-CD20 therapy due to weak and various expression of CD20 in the preponderance of subjects. In contrast, other studies demonstrated that the CD20+ phenotype is associated with patients with t(11,14)(q13;q32) and with shorter survival  and that sporadic responses have been achieved in patients 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%) who express CD20 in plasma cells, but a different mechanism that may render MM refractory to rituximab is the possibility that MM cells express increased levels of complement-inhibiting proteins, such as the presence of the complement regulator CD59 on myeloma makes complement-mediated cytotoxicity inefficacious [16, 20-22]. In addition, Fc-c receptor polymorphism may limit the efficacy of ADCC as a killing mechanism. Finally, the administration of rituximab in MM may cause 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 radiolabeled murine anti-CD20 antibody, which is highly effective in the treatment of low-grade B-cell NHL. It was tested in a single 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 previously treated MM and produces objective responses including CRs .
Available data indicate that progression-free survival of MM patients is improved with IFNα, but although IFNα can have direct cytotoxic action on plasma cells, promote both innate and adaptive immunity, and inhibit angiogenesis, its efficaciousness as an anticancer drug has been limited due to its short circulating half-life and systemic toxicity .
Rossi et al. reported the generation of the first bispecific MoAb-IFNα, designated 20-C2-2b, which comprises two copies of IFNα2b and a stabilized F(ab)2 of hL243 (humanized anti-HLA-DR; IMMU-114) site specifically linked to veltuzumab (humanized anti-CD20). In vitro, 20-C2-2b inhibited eight myeloma cell lines and was more effective than monospecific CD20-targeted MoAb-IFNα or a mixture comprising the parental antibodies and IFNα in all but one (HLA-DR−/CD20−) myeloma line, suggesting that 20-C2-2b should be useful in the treatment of MM. 20-C2-2b displayed greater cytotoxicity against KMS12-BM (CD20+/HLA-DR+ myeloma) compared with monospecific MoAb-IFNα, which targets only HLA-DR or CD20, indicating that all three components in 20-C2-2b could contribute to toxicity .
The cell surface glycoprotein CS1 (CD2 subset 1, CRACC, SLAMF7, CD31, or 19A24), a member of the signaling lymphocyte-activating molecule–related receptor family, is selectively and constantly expressed at high levels on CD138-purified plasma cells from the MM patients (>97%), independent of the presence of metaphase cytogenetic abnormalities or molecular subgroup. In addition, low levels of circulating CS1 are present in myeloma patient sera. Except activated B, NK, CD8+ T cells, and mature dendritic cells with low expression levels, it is not expressed by normal tissues or stem cells [26-28]. Moreover, it regulates NK cell cytolytic activity via recruiting EWS-activated transcript-2 and activating the PI3K/PLCg signaling pathways [29, 30].
Studies propose that CS1 localizes to the uropods of polarized myeloma cells suggesting a possible function for CS1 in mediating adhesion of myeloma cells to bone marrow stroma. CS1 also seems to preserve myeloma cell lines from apoptosis by lowering phosphorylation of ERK1/2, AKT, and STAT as well as regulating pro- and anti-apoptotic pathways .
CS1 may then contribute to MM pathogenesis by increasing MM cell adhesion, clonogenic growth, and tumorigenicity via c-Maf-mediated interactions with BMSCs .
The humanized anti-CS1 MoAb elotuzumab exerts antimyeloma activity in vitro via ADCC mediated by NK cells and does not depend on complement-mediated cytotoxicity .
In fact, elotuzumab caused significant ADCC against MM cells even in the presence of BMSCs. Moreover, it provoked autologous ADCC against primary MM cells resistant to traditional or new drugs including HSP90 inhibitor and bortezomib and, considerably, increased HuLuc63-induced MM cell lysis when pretreated with conventional or new anti-MM therapies .
Administration of elotuzumab causes tumor regression in myeloma xenograft mouse models . Furthermore, combination of elotuzumab with bortezomib significantly increases the in vivo therapeutic efficacy to eradicate patient-derived myeloma cells in a SCID-hu mouse model .
In a phase 1, multicenter, dose-escalation study of elotuzumab in patients with advanced MM, patients with relapsed/refractory MM were treated with intravenous elotuzumab. The most common AEs were cough, headache, back pain, fever, and chills. Adverse events were generally mild to moderate in severity. Nearly 26.5% had stable disease .
In other studies, most AEs attributable to elotuzumab included mild-to-moderate infusion reactions, and the implementation of more aggressive premedication regimens appeared 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 activity in vitro. Pretreatment with dexamethasone or new drugs such as bortezomib, lenalidomide, MEK inhibitors, or Akt inhibitors has been shown to significantly increase elotuzumab-induced ADCC against MM cells [26, 34, 38].
Preliminary results of clinical trials of HuLuc63 in combination with bortezomib or lenalidomide or dexamethasone were reported at the ASH meeting 2009 [39, 40], suggesting that elotuzumab may increase the activity of bortezomib and lenalidomide in treating MM with acceptable toxicity.
Rhee et al. investigated whether the activity of elotuzumab could be increased by bortezomib. In vitro bortezomib pretreatment of myeloma targets significantly increased elotuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), both for OPM2 myeloma cells using NK or peripheral blood mononuclear cells (PBMC) from controls and for primary myeloma cells using autologous NK effector cells. In an OPM2 myeloma xenograft model, elotuzumab in combination with bortezomib revealed significantly increased in vivo antitumor activity .
A phase 1 study evaluated elotuzumab, lenalidomide, and dexamethasone in patients with relapsed or refractory MM. The most frequent grades 3 to 4 toxicities were neutropenia (36%) and thrombocytopenia (21%). Objective responses were obtained in 82% of treated patients. The combination of elotuzumab, lenalidomide, and low-dose dexamethasone was generally well tolerated and showed promising response rates in patients with relapsed or refractory MM .
In phase 1/2 studies in relapsed/refractory MM, elotuzumab and bortezomib were administered. The most frequent grades 3 to 4 adverse events were lymphopenia and fatigue. Two elotuzumab-related serious AEs (chest pain and gastroenteritis) occurred in one patient. An objective response was observed in 48% of evaluable patients and in 67% of three patients refractory to bortezomib. Median time to progression was 9.46 months .
CD138 (syndecan-1) is a heparan sulfate proteoglycan that serves as a receptor for epidermal growth factor (EGF) ligands. Binding of EGF ligands stimulates cell growth .
Moreover, the large extracellular domain of CD138 binds via its heparin sulfate chains to other soluble extracellular molecules, including the fibroblast growth factor and hepatocyte growth factor, and to insoluble extracellular molecules, such as collagen and fibronectin. CD138 also mediates cell–cell adhesion through interactions with heparin-binding molecules. Studies of plasma cell differentiation show that CD138 is a differentiation antigen and a coreceptor for MM growth factors .
Immunohistochemical and flow cytometric analysis of patient MM cells has shown that CD138 is expressed in a large majority of cases. Within the hematopoietic compartment, CD138 expression is confined to normal plasma cells, with no expression on hematopoietic stem cells (HSC), while expression of CD138 on MM cells is significantly higher than on normal plasma cells .
Almost all MM cells, even after exposure to multiple therapies, express the antigen, making it a useful target at any stage of the disease. The extracellular components of CD138 and the heparan sulfate side chains may be shed from the cell surface. Soluble syndecan-1 in serum serves as a prognostic indicator in MM .
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 moiety and MoAb. The nBT062-SMCC-DM1 linkage contains a thioether bond, which is not cleavable by disulfide exchange, whereas the nBT062-SPDB-DM4 and nBT062-SPP-DM1 conjugates contain disulfide linkages, which can be cleaved by disulfide exchange, resulting in liberation of active maytansinoid agent. The anti-CD138 antibody nBT062 is a murine/human chimeric form of B-B4. The observed preclinical antitumor activity of the nBT062–maytansinoid conjugates provides the framework for clinical development of these agents to improve patient outcome in MM .
Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against PBMC from healthy controls. 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 cytotoxicity induced by nBT062–maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062–maytansinoid conjugates blocked adhesion of MM cells to BMSC. The coculture of MM cells with BMSC protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model .
Tassone et al. first reported the potential of tumor targeting with an anti-CD138 antibody–maytansinoid conjugate using the murine parent of the antibody (B-B4) found in BT062. Treatment of CD138-positive cells with B-B4-DM1 significantly reduced cell survival in a dose-dependent manner, while B-B4 antibody alone had little efficacy .
Antitumor activity of B-B4-DM1 was also assessed in MM xenograft studies in mice. Marked tumor regressions were observed upon treatment with B-B4-DM1 in subcutaneous MM models as measured by tumor volume or by fluorescent imaging of green fluorescent protein–expressing tumors. The antitumor activity of B-B4-DM1 was proved in a SCID-hu model of human MM, where patient MM cells proliferate in a human bone chip microenvironment .
Recently, Rousseau et al. reported preliminary biodistribution and dosimetry results obtained in refractory MM patients in a phase 1/2 RAIT study using iodine-131-labeled anti-CD138 (B-B4) monoclonal antibody (MoAb). Four patients with progressive disease were enrolled after three lines of therapy. Grade 3 thrombocytopenia was documented in two cases, and no grade 4 hematologic toxicity was observed. One patient experienced partial response, with 60% reduction in M-spike on serum electrophoresis, and total alleviation of pain, lasting for 1 yr .
CD38 is a 46-kDa type II transmembrane glycoprotein with a short 20-aa N-terminal cytoplasmic tail and a long 256-aa extracellular domain. Functions ascribed to CD38 include receptor-mediated adhesion and signaling events, as well as important activities that contribute to intracellular calcium mobilization. Under normal conditions, CD38 is expressed at relatively low levels on lymphoid and myeloid cells and in some tissues of non-hematopoietic origin. The relatively high expression of CD38 on all malignant cells in MM in combination with its role in cell signaling suggests CD38 as a potential therapeutic 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 applications . Nevertheless, recently, a human anti-CD38 IgG1 HuMax-CD38 (daratumumab) was raised after immunizing transgenic mice (HuMax-Mouse) possessing human, but not mouse, Ig genes. Preclinical works evidenced that HuMax-CD38 was able in killing primary CD38+ CD138+ patient MM cells and a range of MM/lymphoid cell lines by both ADCC and CDC .
De Weers et al. reported the cytotoxic mechanisms of action of daratumumab. Daratumumab induced potent ADCC in CD38-expressing MM-derived cell lines as well as in patient MM cells. Daratumumab stood out from other CD38 MoAbs in its strong ability to induce CDC in patient MM cells. Importantly, daratumumab-induced ADCC and CDC were not affected by the presence of BMSC, suggesting that daratumumab can effectively kill MM tumor cells in a tumor-preserving bone marrow microenvironment. In vivo, daratumumab was highly active and interrupted xenograft tumor growth at low dosing .
Van der Veer et al. investigated the action of lenalidomide combined with daratumumab. Daratumumab-dependent cell-mediated cytotoxicity of purified primary MM cells, as well as of the UM-9 cell line, was significantly enhanced by lenalidomide pretreatment of the effector cells derived from PBMC from healthy individuals. More importantly, they demonstrated a synergy between lenalidomide- and daratumumab-induced antibody-dependent cell-mediated cytotoxicity directly in the bone marrow mononuclear cells of MM patients, indicating that lenalidomide can also potentiate the daratumumab-dependent lysis of myeloma cells by activating the autologous effector cells within the natural environment of malignant cells. Finally, daratumumab-dependent cell-mediated cytotoxicity was significantly up-regulated in PBMC derived from three MM patients during lenalidomide treatment.
The lenalidomide–daratumumab combination appears to be a highly attractive choice because these results demonstrate that lenalidomide significantly synergizes with daratumumab to improve the MM cell lysis .
In a dose-escalation study of daratumumab in patients with MM, the safety profile has been acceptable. In patients with relapsed or refractory MM treated with daratumumab, a marked reduction in paraprotein and bone marrow plasma cells was observed in the higher dose cohorts. This has not previously been demonstrated with a single-agent monoclonal antibody in MM. No unexpected buildup of daratumumab was seen, and in patients treated with 4 mg/kg and upwards, the observed plasma concentrations were close to those predicted. The MTD was not yet established, and the toxicity was manageable .
Finally, MOR202 (MorphoSysAG), a fully human anti-CD38 IgG1 MoAb produced by a human combinatorial antibody library (HuCAL) platform, also efficiently triggers ADCC against CD38+ MM cell lines and patient MM cells in vitro as well as in vivo in a xenograft mouse model .
CD40 is a transmembrane glycoprotein of the tumor necrosis factor (TNF) receptor superfamily that is involved in B-cell activation and the formation of germinal centers . 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 participate in B-cell tumorigenesis via NFjB, ERK, p38 MAPK, and PI3-kinase signaling . CD40L-CD40 signaling also participates in protective tumor immunity. Thus, inhibition of CD40L-CD40 signaling reduces tumor cell proliferation and survival and may disrupt the protective immune state and stimulate immune-mediated antitumor activity in MM [71, 72].
Lucatumumab (HCD122, formerly CHIR-12.12) is a fully human, recombinant IgG1 isotype monoclonal antibody that targets CD40 and inhibits the growth and survival of B-cell malignancies. In contrast to other anti-CD40 monoclonal antibodies that demonstrate agonist activity, lucatumumab is a 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 to reduce B-cell differentiation. Lucatumumab also mediates an antitumor immune response by binding effector cells and inducing cell lysis via antibody-dependent cell-mediated cytotoxicity (ADCC) in B-cell malignancies, including CD40-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 were anemia, chills, hypercalcaemia, and pyrexia (7% each). 43% of patients had stable disease, and one patient (4%) maintained a partial response for 8 months. These findings indicate that single-agent lucatumumab was well tolerated up to 4.5 mg/kg with modest clinical activity in relapsed/refractory MM .
Dacetuzumab is a different humanized anti-CD40 monoclonal antibody with multiple mechanisms of action. Dacetuzumab kills tumor cells via ADCC and phagocytosis and induction of apoptosis through direct signal transduction [77-79].
Previous works have evaluated the pharmacokinetics (PK), safety, and efficacy of dacetuzumab monotherapy in patients with relapsed/refractory MM . Phase 1 data suggest it is well tolerated with no immunogenicity .
Treatment was generally well tolerated. The most common adverse events were fatigue, headache, nausea, and anemia. Although transient decreases in serum and 24-h urine M-protein levels were observed for some patients, no objective responses were reported.
While single-agent dacetuzumab was not highly active in this study, the observed safety profile suggested that testing dacetuzumab in combination with other MM therapies would be possible. Preliminary in vitro results report that combining dacetuzumab with lenalidomide is synergistic, and this combination may produce better response rates. Two trials are underway in patients with relapsed MM to evaluate dacetuzumab in combination with lenalidomide or bortezomib [82, 83].
Preclinical data have indicated that lenalidomide augments the anti-MM efficacy of dacetuzumab, and the combination regimen is undergoing clinical evaluation. A phase 1b study of combination dacetuzumab plus lenalidomide and dexamethasone in subjects with relapsed or refractory MM has produced promising clinical response rates. The regimen was generally well tolerated; fatigue, neutropenia, and thrombocytopenia were the most common adverse events .
CD56 (neuronal cell adhesion molecule) is a membrane glycoprotein from the immunoglobulin superfamily , expressed on muscle cells and neurons. It appears to mediate cell 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 adhesion molecule correlates with the presence of lytic bone lesions in MM and distinguishes myeloma from MGUS and lymphomas with plasmacytoid differentiation .
Several studies have brought to the formation of anti-56 antibodies conjugated to cytotoxic moieties that combine the specificity of antimyeloma-targeting antibodies with highly active antitumor compounds. One such immunoconjugate currently in clinical development is composed of antibodies that target cell surface proteins found on MM cells and are coupled to cytotoxic maytansinoids .
Lorvotuzumab mertansine (IMGN901) is an immunoconjugate composed of a humanized MoAb to CD56 (huN901) conjugated to the maytansinoid N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1), a potent antimicrotubular cytotoxic agent. HuN901-DM1 has significant in vitro and in vivo anti-MM activity at low doses. Target-dependent cytotoxicity was shown in cocultures of CD56+ and CD56− cells. Importantly, adhesion of CD56+ MM cell lines and patient MM cells to BMSCs, which is known to protect MM cells from drug-induced cytotoxicity, did not protect against the specific cytotoxicity of IMGN901. Treatment with IMGN901 in a human MM tumor xenograft model in immune-compromised mice showed that the immunoconjugate was effective in both a minimal and bulky disease setting .
The phase 1 clinical study of huN901-DM1 (BB-10901) in MM patients demonstrated an overall favorable safety profile [93, 94]. There were no hypersensitivity reactions, and no patients developed antibodies to huN901 or DM1. Three patients achieved MR, with reductions in serum M component and urine M component. Eight patients achieved durable stable disease, and two patients remained on treatment for 42 wk. Finally, additive to synergistic activity has been observed in combinations of IMGN901 with lenalidomide, bortezomib or melphalan [95, 96].
The insulin-like growth factor (IGF) signaling system is comprised of the IGF ligands (IGF-1 and IGF-2), the cell surface receptors 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 . IGF-1 produced by the microenvironment induces the activation of several pathways such as phosphatidylinositol-3 kinase (PI-3K)/Akt, Janus kinase (JAK)/signal transducer and activator of transduction 3 (STAT3), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and nuclear factor-kappa B (NF-κB).
In vivo and in vitro, IGF-1 decreases drug sensitivity of MM cells and up-regulates several anti-apoptotic proteins such as A1/Bfl-1, XIAP, and Bcl-2 and causes a reduction in pro-apoptotic proteins (such as caspase 3, caspase 8, caspase 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 interaction, subsequently blocking the stimulation of signalization pathways, especially PI-3K/Akt and NF-κB .
Tagoug et al. analyzed the effect of the combination of the IKK2 inhibitor AS602868 (an anilinopyrimidine derivative and adenosine triphosphatase competitor selected for its inhibitory effect on IKK2 in vitro) and a monoclonal antibody directed against IGF-1R on MM cell lines. They found that anti-IGF-1R potentiated the apoptotic effect of AS602868 in LP1 and RPMI8226 MM cell lines, which express high levels of IGF-1R. Anti-IGF-1R enhanced the inhibitory effect of AS602868 on NF-κB pathway signaling and potentiated the disruption of mitochondrial membrane potential caused by AS602868 [105, 106].
AVE1642 is a humanized version of the murine monoclonal antibody, EM164, raised against the human IGF-1R. It has been shown to bind specifically, with a high affinity, to human IGF-1R, to inhibit IGF-1 binding and receptor activation, and to down-regulate the receptor by internalization and degradation. AVE1642 has been able to delay growth and survival of some cancer cells in vitro, human tumor xenografts in nude mice, and to inhibit proliferation and survival of most MM cells [107, 108].
Moreau et al. reported the results of a phase 1, multicenter, open-label study, made to evaluate the effects of AVE1642 alone and in combination with bortezomib, in patients with relapsed MM. Despite the good toxicity profile of the antibody, the response rates for patients treated with AVE1642 in this study, as a single agent or in combination with 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 with regard to their IgG subclass and pharmacokinetic properties, but do share some similarities. Generally, they have an advantageous toxicity profile, and no dose-limiting toxicities have been described .
The first-in-human study of figitumumab (CP-751,871) was conducted in patients with refractory myeloma. No dose-limiting toxicities (DLTs) were observed, although individual cases of grade 3 adverse events of hyperglycemia and anemia were seen. Of the 27 patients who received both figitumumab and dexamethasone, nine responded .
Finally, dalotuzumab (MK-0646; h7C10), is a recombinant humanized IgG1 MoAb against the IGFR1. Preliminary data from phase I clinical trials suggest that dalotuzumab is safe, well tolerated, and significantly inhibits tumor proliferation. Several clinical trials evaluating dalotuzumab, alone and in combination with other anticancer agents, were ongoing in patients with MM .
Anti-HM1.24 (anti-317) MoAb
The HM1.24 antigen (HM1.24) is a transmembrane protein that has unique topology with two membrane anchor domains: an NH2-terminal transmembrane domain and a glycosylphosphatidylinositol attached to the COOH terminus . HM1.24 (CD317) was originally identified as a cell surface protein differentially overexpressed on MM cells and later was found to be identical to bone stromal cell antigen 2 (BST-2) [119, 120].
A role of HM1.24 in trafficking and signaling between the intracellular and cell surface of MM cells was suggested because it is one of the important activators of NF-κB pathway.
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 and prolongs survival of myeloma-bearing mice in myeloma xenograft mouse models . However, its antimyeloma activity is diminished when the mice are pretreated with anti-Fcγ 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 inhibited tumor growth in both orthotopic and ectopic human MM xenograft models. A phase 1/2 clinical study showed that a humanized anti-HM1.24 MoAb did not cause any serious toxicity when administered to patients with relapsed or refractory MM .
Ishiguro et al. produced defucosylated AHM and evaluated it by performing autologous ADCC assays against primary MM cells from patients. Defucosylated AHM showed significant ADCC activity against three of six primary MM cells in the presence of autologous PBMC, whereas conventional AHM did not. The results indicate that the potency of AHM to induce ADCC against primary MM cells was insufficient, but was significantly augmented by defucosylation .
Tai et al. investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 MoAb with Fc domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased ADCC several folds relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody-dependent cellular phagocytosis (ADCP) by macrophages. NK cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B-dependent CD107a and MM cell lysis, even in the presence of BMSCs. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone 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 of MM .
CD48 is a 47-kD glycophosphatidylinositol-linked glycoprotein that is expressed on mature lymphocytes and monocytes, but not on non-hematopoietic tissues .
Hosen et al. found that CD48 was highly expressed on MM plasma cells. In 22 of 24 MM patients, CD48 was expressed on more than 90% of MM plasma cells at significantly higher levels than it was on normal lymphocytes and monocytes. CD48 was only weakly expressed on some CD34+ hematopoietic stem/progenitor cells and not expressed on erythrocytes or platelets.
Administration of the anti-CD48 MoAb significantly inhibited tumor growth in severe combined immunodeficient mice inoculated subcutaneously with MM cells. Furthermore, anti-CD48 MoAb treatment inhibited growth of MM cells transplanted directly into murine bone marrow. Finally, they demonstrated that the anti-CD48 MoAb did not damage normal CD34+ hematopoietic stem/progenitor cells .
However, a major concern regarding CD48 as a therapeutic target is its broad expression on normal lymphocytes and monocytes, which may induce severe cytopenia and immunosuppression when anti-CD48 MoAb is used as a therapeutic drug. The potential hematologic toxicity of anti-CD48 MoAb should therefore be very carefully tested, and anti-CD48 MoAb may not be suitable for long-term maintenance therapy because of hematologic toxicities. For induction therapy, it could be useful for the eradication of MM plasma cells.
Anti-β2 microglobulin MoAb
β2-microglobulin (β2M) is an 11.6-kDa non-glycosylated polypeptide composed of 100 amino acids. It is part of the MHC class I molecule on the cell surface of nucleated cells. Its best characterized function is to interact with and stabilize the tertiary structure of the MHC class I α-chain .
High levels of serum β2M are present in hematologic malignancies, including MM, and correlate with a poor outcome regardless of a patient's renal function [131, 132].
Anti-β2M MoAbs have been developed recently . Several studies demonstrated that anti-β2M MoAbs have remarkably strong tumoricidal activities to kill all examined myeloma, including tumor cell lines and primary CD138+ malignant plasma cells isolated from subjects with MM, and β2M+/HLA-ABC+ hematologic malignant cells. Furthermore, they provoke a direct induction of tumor cell death without the need for exogenous immunologic effector cells and/or molecules, and they have capacity to kill chemotherapy-refractory myeloma. They demonstrated therapeutic efficacy in vivo in xenograft mouse models of myeloma and kill myeloma cells in the presence of BMSCs. Although the mechanisms of its action in myeloma cell death need to be further investigated, evidence has shown that anti-β2M MoAbs directly induce tumor cell apoptosis without immunologic effector mechanisms .
β2M/MHC class I complex has been shown to operate as an important signal-transducing molecule, which is involved in responses ranging from anergy and apoptosis to cell proliferation and IL-2 production [134, 135]. Earlier studies showed that cross-linking of β2M/MHC class I induces a rise of intracellular free calcium concentration and activation of STAT3 and/or JNK through phosphorylation of a signaling motif at tyrosine 320 residue in the cytoplasmic domain of MHC class I α-chain [136, 137]. It was evidenced that binding of anti-β2M MoAbs to myeloma cells results in internalization and down-modulation of surface β2M/MHC class I molecules and induction of myeloma cell apoptosis . Knockdown of surface β2M by specific small interference RNAs (siRNAs) significantly abrogates the MoAb-induced tumor cell apoptosis. As a result, cross-linking of β2M/MHC class I molecules by the MoAbs activates JNK and inhibits PI3K/Akt and ERK, leading to compromised mitochondrial integrity, cytochrome c release, and activation of the caspase-9 cascade . However, the mechanisms underlying the MoAb-mediated binding to and cross-linking of surface β2M/MHC class I molecules, and transduction of apoptotic signals to cells needs further investigation .
Nevertheless, IgG anti-β2M MoAbs induced apoptosis in up to 90% of cells in a 48-h culture in all tested human myeloma cell lines (HMCLs) and primary myeloma cells from patients. Anti-β2M MoAb-induced apoptosis in myeloma cells was not blocked by soluble β2M, IL-6, or other myeloma growth factors and was stronger than apoptosis observed with chemotherapy drugs currently used to treat MM (e.g., dexamethasone) .
Finally, Cao et al. hypothesized that IgM anti-β2M MoAbs might have stronger apoptotic effects because of their pentameric structure. Compared with IgG MoAbs, IgM anti-β2M MoAbs exhibited stronger tumoricidal activity in vitro against myeloma cells and in vivo in a human-like xenografted myeloma mouse model without damaging normal tissues. IgM MoAb-induced apoptosis is dependent on the pentameric structure of the MoAbs. Disrupting pentameric IgM into monomeric IgM significantly reduced their ability to induce cell apoptosis. Monomeric IgM MoAbs were less efficient at recruiting MHC class I molecules into and exclusion of cytokine receptors from lipid rafts, and at activating the intrinsic apoptosis cascade .
CD70 is a member of TNF family. Interaction of CD70 and its ligand CD27 modulates the expansion and differentiation of effector and memory T-cell populations  and promotes B-cell expansion and plasma cell differentiation . CD70 is only transiently expressed on activated B cells, T cells, mature dendritic cells, and thymic medulla stromal cells, but it is not expressed in other normal, non-hematopoietic tissues . However, aberrant CD70 expression in tumor cells has been reported in NHL, B-cell lymphocytic leukemias, Waldenström macroglobulinemia, and MM .
SGN-70, a humanized MoAb specific to CD70, has been developed . SGN-70 exhibits potent antimyeloma activity in vitro and significantly prolonged the survival of tumor-bearing mice in vivo. This MoAb induces Fc-mediated effector functions, such as ADCC, complement fixation, and CDC.
CD74 is an integral membrane protein that was described in 1970 as an invariant chain (Ii) of the major histocompatibility complex II (MHC II) in both humans and mice [145, 146]. It may interact with macrophage migration inhibitory factors that are critical mediators of the host defense and is involved in both acute and chronic response . Recent studies have shown that CD74 is frequently expressed in MM . Malignant plasma cells from 80% of MM patients and from majority of myeloma cell lines express CD74 mRNA and protein.
Moreover, CD74 protein is expressed in more than 85% of NHL and CLL, while it shows limited expression in normal human tissues .
Milatuzumab is a humanized anti-CD74 MoAb that shows selective binding and rapid internalization into CD74-positive cancer cells. Milatuzumab causes growth inhibition and induction of apoptosis in CD74-expressing MM cell lines when cross-linked with an anti-human immunoglobulin G secondary antibody . Moreover, milatuzumab demonstrated promising therapeutical activity in a CAG-SCID mouse model ofMM when used alone or in combination with doxorubicin, dexamethasone, bortezomib, or lenalidomide. Treatment of CAG and KMS-11 MM tumors with milatuzumab, bortezomib, and combination of both agents showed a significantly greater efficacy of milatuzumab than that of bortezomib for treatment of these tumors. Additionally, the combination of both agents was even more efficacious [150, 151].
In a phase I trial, milatuzumab showed no severe adverse effects in patients with relapsed/refractory MM, and it stabilized 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 two previous standard therapies. Milatuzumab was administered twice weekly for 4 wk. There was a reported dose-limiting toxicity infusion reaction and severe adverse effects (SAEs), which included a case of bacterial meningitis, fever, unexplained hemoglobin drop, cord compression confusion, hypercalcemia, and thrombocytopenia .
Antagonistic targeting of CD74 by milatuzumab could remove autocrine prosurvival signals and reinstate Fas-mediated apoptotic signaling.
Additional MoAbs are directed against a variety of further MM cell targets including HLA-DR by 1D09C3  and HLA class I by 2D7-DB .
NK cells play a critical role in ADCC to lyse tumor target cells via therapeutic monoclonal antibodies, inhibitory-cell killer immunoglobulin-like receptors (KIRs) negatively regulate NK cell-mediated killing of HLA class I–expressing tumors, and MoAbs targeting KIR might prevent their inhibitory signaling leading to enhanced ADCC. A new fully human 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 cytotoxicity against autologous MM tumor cells in vitro and appears safe in an ongoing phase I clinical trial . A multicenter, open-label phase IIa clinical trial has started to evaluate IPH 2101 as a single agent in patients with stable measurable MM after induction therapy. Another phase II clinical trial to assess the potential of lenalidomide combined with 1-7F9 will be initiated in patients with MM .
KIRs are receptors expressed on NK cells and a subset of T cells and function as key regulators of NK cell activity . Several studies are currently underway in smoldering and first relapse MM (NCT01222286, NCT01217203, NCT00999830, NCT01248455), and safety and tolerability results are expected for a phase 1 study in relapsed or refractory MM (NCT00552396).
Kimura et al. demonstrated that HLA class I molecules are overexpressed in MM cells and that a recombinant single-chain Fv diabody against this molecule efficiently cross-links HLA class I and induces rho-mediated actin aggregation, which leads to MM cell death without effector function [154, 159].
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 the immunoglobulin superfamily and are involved in lymphocyte activation [160, 161].
The SLAM family member CD229, which has been shown to interact with its intracellular adapter protein Grb2 in a phosphorylation-dependent manner, showed the strongest overexpression/phosphorylation in all myeloma cell lines .
Atanackovic et al. identified CD229 as the most strongly over-expressed/phosphorylated immunoreceptor in myeloma cell lines. Overexpression was further demonstrated in the CD138-negative population, which has been suggested to represent myeloma precursors, as well as on primary tumor cells from myeloma patients. Accordingly, CD229 staining of patients' bone marrow samples enabled the identification of myeloma cells by flow cytometry and immunohistochemistry. Down-regulation of CD229 led to a reduced number of viable myeloma cells and clonal myeloma colonies and increased the antitumor activity of conventional chemotherapeutics. Targeting CD229 with a monoclonal antibody resulted in complement- and cell-mediated lysis of myeloma cells .
Anti-GM-2 ganglioside MoAb
GM-2 is a ganglioside expressed on MM cells. A humanized anti-GM-2 MoAb, BIW-8962, has demonstrated in vitro killing of MM cell lines and in vivo effectiveness in mouse xenograft models, with ADCC and CDC the most prominent cytotoxic mechanisms. BIW-8962 is being evaluated as monotherapy in a phase 1/2 study for patients with relapsed/refractory MM .
CD200 is a highly conserved transmembrane glycoprotein expressed on a wide range of cell types; however, expression of the receptor for CD200 (CD200R1) is apparently confined to antigen-presenting cells of myeloid lineage and certain T-cell populations and is thought to deliver inhibitory signals. Several studies have shown that CD200 imparts an immunoregulatory signal through CD200R, leading to the suppression of T-cell-mediated immune responses. CD200-deficient mice have a compromised capacity to down-regulate the activation of antigen-presenting cells .
Moreaux et al. identified that CD200 was expressed in malignant plasma cells in 78% of newly diagnosed MM patients. The expression of the CD200 gene by MM cells has been found to be a predictor of poor prognosis in patients with MM .
ALXN6000 is a humanized anti-CD200 MoAb that is currently being evaluated in a phase 1/2 study in patients with MM or B-cell CLL (NCT00648739), with results expected in the near future.
Anti-CD 54 (ICAM-1) MoAb
Intercellular adhesion molecule-1 (ICAM-1) mediates adhesion of myeloma cells to BMSCs and thereby participates to cell adhesion–mediated drug resistance. ICAM-1 is highly expressed on the plasma cell surface in the majority of MM subjects, but is also expressed on normal cell types, including epithelial cells, endothelial cells (ECs), fibroblasts, and several types of leukocytes . Expression of ICAM-1 on myeloma cells increases after chemotherapy, and high levels of ICAM-1 predict poor response to therapy in chemo-naive patients. Moreover, macrophages preserve myeloma cells from melphalan or dexamethasone-induced apoptosis in vitro. Zheng et al. found that macrophage-mediated myeloma drug resistance was also seen with purified macrophages from myeloma patients' bone marrow in vitro and was confirmed in vivo using the human myeloma-SCID mouse model. They showed that ICAM-1/CD18 played an important role in macrophage-mediated myeloma cell drug resistance, as blocking antibodies against these molecules or genetic knockdown of ICAM-1 in myeloma cells repressed macrophages' ability to protect myeloma cells.
BI-505 is a fully human immunoglobulin G1 antibody specific to ICAM-1 that kills myeloma cells in vitro through induction of ADCC and apoptosis. In addition, BI-505 inhibits myeloma tumor growth in mice. On the basis of these data, a phase 1 dose-escalation study is currently recruiting relapsed/refractory myeloma patients to evaluate efficacy and toxicity of single-agent BI-505 .
The Ku heterodimer is made up of two subunits of approximately 86 and 70 kDa in higher eukaryotes, which binds to DNA. Identified in 1981 from Japanese patients with scleroderma–polymyositis overlap syndrome, Ku has now been found to play major roles in many cellular processes . First of all, Ku86 in association with Ku70 and DNA protein kinase C plays a critical role in DNA repair especially in non-homologous end joining where the lack of functional Ku proteins in the cell typically results in genomic instability and hypersensitivity to DNA damage as well as an increased likelihood of tumor development and immunodeficiency [170-173]. Moreover, Ku86 has been discovered to translocate to the cell surface of MM cells upon CD40L treatment and also mediate the binding of MM cells to fibronectin and BMSC. Most importantly, the MoAb 5E2 directed against Ku86 were seen to induced apoptosis of MM cells [174-176].
Liew et al.  found good-affinity antibodies against Ku86, and they can be used for further studies on MM and form the basis for further development as anticancer therapeutic agents.
MoAbs Targeting components of bone marrow milieu
Autocrine and paracrine IL-6 have been seen to play a crucial role in growth and survival of MM cells within the BM milieu . In BM microenvironment, IL-6 is predominantly produced by BMSCs, mediating MM cell growth and preventing apoptotic cell death. IL-6 stimulates at least three different signaling pathways; Ras/MEK/ERK cascade, JAK2/signal transducer and activator of transcription (STAT-3) cascade, and PI3K/Akt cascade. Importantly, IL-6 protects against apoptotic cell death induced by a variety of agents. In addition, IL-6 also controls expression of various other key growth in MM subjects. For example, IL-6 plays an important role in the transcriptional regulation of myeloid cell leukemia (Mcl)-1, an anti-apoptotic B-cell lymphoma-2 family member, a critical mediator of MM cell survival, and tightly regulated by the proteasome . Therefore, down-regulation of IL-6 signaling would also sensitize MM cells to proteasome inhibitor-mediated apoptosis by interfering with the induction of the HSP response and Mcl-1. IL-6 is also a potent osteoclast activating factor (OAF) for human osteoclast precursors [181-186], while clinical studies have revealed that increased serum IL-6 concentrations in patients are associated with advanced stages of MM and short survival in patients. Therefore, blocking IL-6 signaling is a potential therapeutic strategy for cancer .
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 in vitro anti-MM activity. However, their in vivo and clinical effectiveness remains ambiguous. For example, in a phase I study, using a mouse–human chimeric monoclonal anti-IL6 antibody, none of the MM patients achieved a response .
Table 2. Monoclonal antibodies targeting components of bone marrow milieu
However, although the clinical activity of single-agent anti-IL-6/IL-6R in MM patients has been limited, any clinical studies of the anti-IL-6 monoclonal antibody CNTO 328 have 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, one in combination with dexamethasone and another with bortezomib, have shown evidence of encouraging activity .
Voorhees et al. evaluated whether CNTO 328 could enhance the apoptotic activity of dexamethasone (dex) in preclinical models of myeloma. CNTO 328 potently increased the cytotoxicity of dex in IL-6-dependent and IL-6-independent HMCLs, including a bortezomib-resistant HMCL. Isobologram analysis revealed that the CNTO 328/dex combination was highly synergistic. Addition of bortezomib to CNTO 328/dex further enhanced the cytotoxicity of the combination. Although CNTO 328 alone induced minimal cell death, it potentiated dex-mediated apoptosis, as evidenced by increased activation of caspase-8, caspase-9, and caspase-3, annexin V staining, and DNA fragmentation. The ability of CNTO 328 to sensitize HMCLs to dex-mediated apoptosis was preserved in the presence of human BMSC .
Hunsucker et al. reported that the combination of siltuximab and melphalan attenuated cell proliferation in an additive to synergistic manner and enhanced apoptosis in HMCLs. This increased cell death correlated with enhanced Bak activation, and siltuximab also inhibited IL-6 activation of the prosurvival PI3-K/Akt signaling pathway. Importantly, the siltuximab/melphalan combination was also effective in patient-derived myeloma samples and partially overcame melphalan resistance .
The anti-interleukin-6 receptor monoclonal antibody tocilizumab (TCZ) was reported to inhibit in vitro proliferation of cloned and freshly isolated myeloma cells from patients with advanced MM . In addition, Nishimoto et al.  reported the first 2 cases of refractory MM treated with TCZ, noting that it improved fever and systemic edema and also stabilized the monoclonal protein levels.
Matsuyama et al. reported a patient with rheumatoid arthritis (RA) and smoldering MM, in whom TCZ improved RA symptoms and also stabilized the serum levels of monoclonal IgA .
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 monotherapy in MM patients who are not candidates for, or who have relapsed after SCT .
In addition, NRI, another receptor inhibitor of IL-6 genetically engineered from tocilizumab, is under preclinical evaluation . An adenovirus vector encoding NRI was administered to mice intraperitoneally (i.p.) and monitored for the serum NRI level and growth reduction property on the xenografted 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 transient tumor cytostasis and reduction in toxicities from IL-6 .
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 was demonstrated. 81.3% of patients exhibited a response, with a complete response seen in 37.5% of patients without any toxic or allergic reactions.
A high-affinity fully human version of BE-8, OP-R003-1 (or 1339, Azintrel), was studied. It enhanced cytotoxicity induced by dexamethasone, as well as bortezomib, lenalidomide, and perifosine, in a synergistic fashion. Importantly, Azintrel also blocked bone turnover in SCID-hu mouse model of MM, providing an additional rationale for its use in MM .
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].
One key compartment of the MM microenvironment is the vascular niche. The role of angiogenesis in hematologic malignancies is now well established. VEGF within the MM BM microenvironment induces growth, survival as well as migration of MM cells in an autocrine manner via VEGFR-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 for plasma cells, including VEGF, FGF-2, MMP-2, as well as MMP-9. Compared with healthy human umbilical vein EC (HUVEC), MMECs secrete higher amounts of the CXC chemokines (e.g., IL8, SDF1-α, MCP-1), which act in a paracrine manner to mediate plasma cell proliferation and chemotaxis. Moreover, MM cells and BBMSC prolong survival of ECs both by increased secretion of EC survival factors, such as VEGF, and by decreased secretion of anti-angiogenic factors [183, 207-210]. Research on angiogenesis 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 . Anti-VEGF Abs were active alone and in combination with radiation in earlier preclinical studies . It is currently being studied clinically in many diseases as systemic amyloidosis and MM . NCI's Cancer Therapy Evaluation Program is sponsoring a phase II study of bevacizumab plus thalidomide in MM .
Attar-Schneider et al. explored the efficacy of anti-VEGF treatment with bevacizumab directly on MM cells. They showed that blocking VEGF is detrimental to the MM cells and provokes cytostasis. This was evidenced in MM cell lines, as well as in primary BM samples (BM MM). Utilizing a constitutively Akt-expressing MM model, they showed that the effect of bevacizumab on viability and eIF4E status is Akt dependent .
Somlo et al. tested the efficacy of bevacizumab alone and in combination with thalidomide in MM patients with a phase II prospective randomized/stratified trial. Toxicities were mild. Among those who received bevacizumab alone, one patient – with the greatest expression of VEGF on MM cells – achieved stable disease for 238 d, but the median EFS for the cohort was only 49 d .
The epidermal growth factor receptor (EGFR) is a member of the ErbB family of transmembrane tyrosine kinase receptors and contributes to malignant cell survival and proliferation .
Upon ligand binding, the EGFR dimerizes in hetero- or homodimers, which results in the activation of an intrinsic tyrosine kinase and the initiation of activating signaling cascades: the Ras and MAPK pathways, and the PI-3K and protein kinase B (Akt) pathways. In addition to activating the pathways that stimulate cell proliferation and cell survival, recent evidence shows that the EGFR might directly act as a transcription factor upon activation .
Cetuximab is a chimeric human–murine monoclonal antibody that binds competitively and with high affinity to the EGFR. Binding of the antibody to the EGFR prevents stimulation of the receptor by endogenous ligands and results in inhibition of cell proliferation and angiogenesis and enhanced apoptosis. Binding of cetuximab to the receptor also results in internalization of the antibody–receptor complex, which leads to an overall down-regulation of EGFR expression .
Cetuximab is approved for the treatment of metastatic colorectal cancer and relapsed/metastatic head-and-neck-cancer and other epithelial malignancies [220, 221]. Recent data indicate that the EGFR is expressed on the malignant plasma cells of MM and on cells of the MM microenvironment. Moreover, MM cells coexpress the EGFR ligands amphiregulin and heparin-binding EGF-like growth factor (HBEGF), and inhibition of EGFR ligand signaling induces MM cell apoptosis [222, 223].
Böll et al. showed an activity of cetuximab as single agent in a patient with relapsed MM. Cetuximab was administered intravenously. After the initiation of cetuximab treatment, they measured disease stabilization as measured by IgG and serum-free kappa light chains in serum and urine. After a total of 44 wk, they stopped cetuximab treatment, and they measured an increase in both IgG and kappa light chains in serum and urine .
At the cytogenetic level, the MM genome is recognized as being complex. The study of chromosomal translocations generated by aberrant class switch recombination shows that several oncogenes, including fibroblast growth factor receptor 3 (FGFR3), are placed under the control of the strong enhancers of the heavy chain Ig (IGH) loci, leading to their deregulation .
Overexpression of FGFR3 has been implicated in the specific changes in gene expression observed in plasma cells as a result of the t(4;14) translocation. Microarray analysis of patient samples with the t(4;14) translocation identified a specific genetic signature characterized by perturbations in the expression of several other genes. A small proportion of patients with the t(4;14) translocation also have FGFR3-activating mutations, including A1157G, A1987G, A761G, and G1138A; however, overall these mutations are rare, with a frequency of approximately 5% among t(4;14) patients .
As a part of the translational effort, new drugs that inhibit oncogenic proteins overexpressed in defined molecular subgroups of the disease, such as FGFR3 and MMSET in t(4;14) MM, are currently being developed .
MFGR1877A is a human IgG1 monoclonal antibody that binds to FGFR3 and is being investigated as a potential therapy for relapsed/refractory FGFR3+ MM. Kamath et al. characterized the PK of MFGR1877A in mouse, rat, and monkey. PK of MFGR1877A was determined in athymic nude mice, Sprague–Dawley rats, and cynomolgus monkeys after administration of single intravenous doses. The antitumor efficacy in mice bearing human tumor xenografts was used in conjunction with inhibitory activity in cell proliferation assays. Doses ranging from 2 to 3 mg/kg weekly to 6–10 mg/kg every 4 wk were predicted to achieve the target exposure in ≥90% of MM patients .
MoAbs Targeting Tumor–BMSC Interaction
MM cells are highly dependent on the BM microenvironment for growth and survival through interactions particularly with BMSCs and osteoclasts, which secrete important MM growth factors. Thus, MoAbs designed to block the binding of MM cell growth and survival factors to their cognate receptors have been under intensive development (Table 3).
Table 3. Monoclonal antibodies targeting tumor–bone marrow stromal cell interaction
Moreover, in addition to therapy directed at MM cells and tumor-promoting interactions, some efforts have been devoted to MoAb therapy directed against the development of complications; to date, these efforts have been restricted to the suppression of myeloma-related bone disease.
Denosumab is an investigational fully human MoAb with high affinity and specificity for RANKL that mimics the natural bone-protecting actions of OPG . Although denosumab was recently approved to treat osteoporosis and prevent the skeletal-related events in patients with bone metastases from solid tumors [230, 231] in the United States and Europe, it is still undergoing phase III clinical trials of its efficacy in treating MM-induced bone disease.
A phase 1 clinical trial in patients with MM or breast cancer with bone metastases showed that following a single s.c. dose of denosumab, levels of urinary and serum N-telopeptide decreased within 1 d, and this reduction lasted through 84 d at the higher denosumab doses .
The most commonly reported adverse events after denosumab administration in patients with MM were fatigue, anemia, upper respiratory tract infection, and headache . In addition, a case of ONJ in a patient who had received denosumab was reported .
Larger trials are ongoing to investigate the effect of denosumab for the treatment of cancer-induced bone disease , and a recent in silico investigation supports the idea that denosumab represents a convenient alternative to pamidronate in the treatment of MM-induced bone disease .
Dickkopf-1 (DKK1), a soluble inhibitor of Wnt/β–catenin signaling required for embryonic head development, regulates Wnt signaling by binding to the Wnt coreceptor lipoprotein-related protein-5 (LRP5)/Arrow. LRP5 mutations causing high bone mass syndromes disrupt DKK1-mediated regulation of LRP5. Forced overexpression of Dkk1 in osteoblasts causes osteopenia, disruption of the HSC niche, and defects in HSC function. Dkk1 also inhibits fracture repair. Studies suggest that DKK1 activation in osteoblasts is the underlying cause of glucocorticoid- and estrogen deficiency–mediated osteoporosis and at least partially underlies the teratogenic effects of thalidomide on limb development.
DKK1 has been implicated in the osteolytic phenotypes of MM. Preclinical studies have shown that neutralizing DKK1/Dkk1 and/or enhancing Wnt/β–catenin signaling may prove effective in treating bone pathologies .
The effect of anti-DKK1 MoAb on bone metabolism and tumor growth in a SCID-rab system has been evaluated. The implants of control animals showed signs of MM-induced resorption, whereas mice treated with anti-DKK1 antibodies blunted resorption and improved the bone mineral density of the implants. Histologic examination revealed that myelomatous bones of anti-DKK1-treated mice had increased numbers of osteocalcin-expressing osteoblasts and reduced number of multinucleated TRAP-expressing osteoclasts. The bone anabolic effect of anti-DKK1 was associated with reduced MM burden .
A different anti-DKK1 agent is BHQ880 [239, 240]. Although BHQ880 had no direct effect on MM cell growth, BHQ880 increased osteoblast differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. Furthermore, in a SCID-hu murine model of human MM, BHQ880 treatment led to a significant increase in osteoblast number, serum human osteocalcin level, and trabecular bone. A preliminary result from a phase I/II trial in MM where BHQ880 was given was well tolerated when given in combination with zoledronic acid.
Finally, Dickkopf-1 (DKK1), broadly expressed in myeloma cells but highly restricted in normal tissues, together with 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 can control myeloma growth or even eradicate established myeloma in vivo. They used DKK1-DNA vaccine in the murine MOPC-21 myeloma model, and the results showed that active vaccination using the DKK1 vaccine not only was able to protect mice from developing myeloma, but also was therapeutic against established myeloma. Mechanistic studies revealed that DKK1 vaccine elicited a strong DKK1- and tumor-specific CD4+ and CD8+ immune responses, and treatment with B7H1 or OX40 Abs significantly reduced the numbers of IL-10-expressing and Foxp3+ regulatory T cells in vaccinated mice .
Anti-activin A MoAb
Activin A is a TGF-β superfamily member most commonly associated with embryogenesis and gonadal hormone signaling . In addition, activin A is involved in bone remodeling with growth stimulatory effects on osteoclasts (Ocs) . Activin A inhibits osteoblasts (OB) differentiation by stimulating SMAD2 activity and inhibiting distal-less homeobox (DLX)-5 expression. More importantly, inhibition of activin A signaling rescued MM-induced OB impairment in vitro and in vivo while reducing MM burden in a humanized myeloma model. Malignant plasma cells disrupt the normal regulatory pathway of bone homeostasis by inducing BMSC secretion of activin A via JNK pathway .
Activin can be targeted by a chimeric antibody RAP-011, derived from the fusion of the extracellular domain of activin receptor IIA and the constant domain of the murine IgG2a. ACE-011, a novel bone anabolic agent currently in a phase 2 clinical trial in MM, is a protein therapeutic based on the activin receptor IIA. In MM, an ongoing multicenter phase 2 trial is conducted in patients who are treated with melphalan, prednisone, and thalidomide. Preliminary results show clinical significant increases in biomarkers of bone formation, improvement in skeletal metastases, and decreases in bone pain as well as antitumor activity. Moreover, ACE-011 has potential as a novel therapy for chemotherapy-induced anemia and may be an effective alternative to erythropoietin (EPO)-based treatments .
B-cell-activating factor (BAFF) is a TNF superfamily member and produced in the bone marrow microenvironment by monocytes, osteoclasts, and neutrophils. Recently, BAFF was recognized as new survival factors for MM . In addition to BMSCs, osteoclasts produce these factors to support MM cells in the BM microenvironment .
Binding of BAFF to the three different TNF-R-related receptors, TACI, BCMA, and BAFF-R, triggers activation of NF-κB, PI3K, and MAPK pathways, resulting in survival and dexamethasone resistance of myeloma cells [248, 249].
BAFF also enhances adhesion of myeloma cells to BMSC . In a mouse model, an anti-BAFF-neutralizing antibody had antimyeloma activity and inhibited osteoclastogenesis.
Atacicept acts as a decoy receptor by binding to and neutralizing soluble BAFF and APRIL and preventing these ligands from 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 or relapsed MM or active, progressive Waldenström macroglobulinemia [251, 252]. Atacicept was well tolerated and showed clinical and biological activity consistent with its mechanism of action. TACI was expressed heterogeneously among patient MM cells, which may explain promising results for the treatment of TACIhigh MM cells in a trial for atacicept .
On the basis of these results, a phase 1 study is currently enrolling relapsed/refractory myeloma patients to evaluate the 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 a potential target for therapeutic intervention. BCMA binds to two ligands that promote tumor cell survival, a proliferation inducing ligand (APRIL) and B-cell activating factor. To selectively target BCMA for plasma cell malignancies, Ryan et al. developed antibodies with ligand-blocking activity that could promote cytotoxicity of MM cell lines as naked antibodies or as antibody–drug conjugates. They showed that SG1, an inhibitory BCMA antibody, blocks APRIL-dependent activation of NF-κB in a dose-dependent manner in vitro. Cytotoxicity of SG1 was assessed as a naked antibody after chimerization with and without Fc mutations that enhance FcgammaRIIIA binding. The Fc mutations increased the antibody-dependent cell-mediated cytotoxicity potency of BCMA antibodies against MM lines by approximately 100-fold with a ≥2-fold increase in maximal lysis. As an alternative therapeutic strategy, anti-BCMA antibodies were endowed with direct cytotoxic activity by conjugation to the cytotoxic drug, monomethyl auristatin F. The most potent BCMA antibody–drug conjugate displayed IC(50) values of ≤130 pmol/L for three different MM lines .
Other potential targets
Anti-TRAIL-R1 R2 MoAb
The TNF family members Fas ligand and TRAIL induce apoptosis upon binding to the death receptors Fas, and TRAIL receptor 1(TRAIL-R1; DR4), or TRAIL receptor 2 (TRAIL-R2; DR5), respectively. TRAIL also binds to the decoy receptors DcR1 and DcR2 and to the soluble decoy receptor osteoprotegerin (OPG), which is produced by osteoblasts and stromal MoAbs activating death receptors [255-258] (Table 4).
Table 4. Other potential targets of monoclonal antibodies
TRAIL-R1 and TRAIL-R2 antimyeloma activity was observed for the fully human agonistic antibody directed against TRAIL-R1 (mapatumumab, HGS-ETR1) and to a lesser 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 MM cell lines are resistant to either antibody .
Importantly, the antimyeloma action of anti-DR4 and anti-DR5 antibodies was not affected by the presence of cells of the bone marrow microenvironment, whereas these cells protected myeloma cells against TRAIL-induced apoptosis . This protective effect was at least partly mediated by OPG.
Bortezomib treatment is associated with the up-regulation of TRAIL and its receptors in B-CLL and lymphoma cells . Furthermore, blockage of TRAIL-R1 and TRAIL-R2 expression using RNA interference, which prevents TRAIL apoptotic signaling, inhibited proteasome inhibitor-induced apoptosis.
Based on enhanced cytotoxicity when combining mapatumumab with bortezomib in preclinical experiments , a randomized phase II study was recently started comparing TRM-1 plus bortezomib versus bortezomib alone in patients with relapsed or refractory MM .
Indeed, combined bortezomib plus mapatumumab treatment resulted in enhanced myeloma cell killing when compared to either agent alone, in both wild-type and bortezomib-resistant cells. In contrast to the preclinical data, a randomized phase 2 study in relapsed/refractory myeloma showed that addition of mapatumumab to bortezomib treatment did not increase response rate or progression-free survival compared with bortezomib alone .
PD-L1 (also known as B7-H1 or CD 274) is a B7 family member and is the ligand for PD-1 (programmed death-1), a member of the B28 family. PD-L1 interacts with PD-1 and an unknown receptor on T cells and can inhibit T-cell activation and cytotoxic T-lymphocytes-mediated lysis .
PD-L1 overexpression appears as a possible mechanism for tumors to avoid the host' immune response, and a way of improving antitumor activity of NK cells against myeloma cells is by modulating the PD-1/PD-L1 axis, which down-regulates the immune response. PD-1 is not present on NK cells from healthy donors, but can be up-regulated by exogenous IL-2. In contrast, PD-1 is present on NK cells from patients, and interaction with PD-L1 on myeloma cells inhibits NK cell function. PD-L1 is expressed on plasma cells from the majority of myeloma patients, but only rarely on plasma cells from MGUS patients and not on normal plasma cells . CT-011 is a humanized anti-PD-1 MoAb, which enhanced NK cell function against myeloma tumor cells through increased NK cell trafficking, improved immune complex formation between myeloma cells and NK cells, and increased NK cell cytotoxicity.
Lenalidomide not only activates NK cells, but also down-regulates PD-L1 expression on myeloma cells and synergized with CT-011 in the activation of NK cells and subsequent killing of myeloma cells. Apart from effects on NK cells, blockade of the PD-1/PD-L1 interaction with anti-PD-L1 MoAbs enhanced myeloma-specific T-cell immunity in vitro and in vivo .
Furthermore, CT-011 also enhanced T-cell responses to autologous dendritic cell/myeloma fusion vaccines . This may be explained by augmented expression of PD-1 on T cells from myeloma patients when compared to healthy controls.
On the basis of these data, a phase 1 clinical trial was started to study the action of CT-011 in patients with advanced hematologic malignancies including myeloma patients. CT-011 was demonstrated to be well tolerated with evidence of antitumor activity. Treatment with CT-011 was accompanied with an elevated percentage of peripheral blood CD4-positive T cells.
The combination of CT-011 with an IMiD may further enhance host antitumor immunity and warrants further investigation. Alternatively, the immune response to myeloma may be enhanced by anti-PD-L1 antibodies such as MDX-1105.
BMSCs as well as extra cellular matrix (ECM) proteins laminin, microfibrillar collagen type IV, and fibronectin are strong adhesive components for MM cells via interaction of a variety of integrins including integrin-a4 (also CD49d, a subunit of CD49d/CD29 or very-late antigen-4; VLA-4) .
A marked increase in BM angiogenesis in MM was found to correlate with VLA-4 expression in plasma cells isolated from patients with active MM. The ability of bortezomib to overcome cell adhesion–mediated drug resistance (CAM-DR) (vincristine and dexamethasone) is, at least in part, due to down-regulation of VLA-4 expression .
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 antibody reduced MM growth, IgG2b production, and associated osteoclastic osteolysis in 5TGM1 murine MM cells both in vitro and in vivo .
Podar et al. evaluated the therapeutic potential of the new-in-class-molecule selective-adhesion molecule (SAM) inhibitor natalizumab, a recombinant humanized IgG4 monoclonal antibody, which binds integrin-a4, iMM .
Natalizumab, but not a control antibody, inhibited adhesion of MM cells to non-cellular and cellular components of the microenvironment as well as disrupted the binding of already adherent MM cells. Consequently, natalizumab blocked both the proliferative effect of MM–BMSC interaction on tumor cells and vascular endothelial growth factor (VEGF)-induced angiogenesis in the BM milieu. Moreover, natalizumab also blocked VEGF- and IGF-1-induced signaling sequelae triggering MM cell migration. Moreover, natalizumab inhibited tumor growth, VEGF secretion, and angiogenesis in a human severe combined immunodeficiency murine model of human MM in the human BM microenvironment.
Importantly, natalizumab not only blocked tumor cell adhesion, but also chemo-sensitized MM cells to bortezomib, in an in vitro therapeutically representative human MM–stroma cell coculture system model .
Anti-CD11C1 (antikininogen) MoAb
Sainz et al. used two cell lines (B38 and C11C1) derived from P3X63Ag8 myeloma cells. The new cell lines were implanted subcutaneously in the strain of origin (Balb/c mice) and used as a model to monitor the effects of C11C1 MoAb to kininogen (HK). They assessed their behavior by intraperitoneal and subcutaneous implantation, by implanting them together and by treating B38-MM with purified MoAb C11C1. They found that MoAb C11C1 inhibits its own tumor growth in vivo and slows down B38-MM growth rate both when MM is implanted together and when MoAb C11C1 is injected intraperitoneally. MAb C11C1-treated MM showed decreased MVD and HK binding in vivo without FGF-2, B1R, or B2R expression changes .