Immunotherapy for the treatment of multiple myeloma

Critical Reviews in Oncology/Hematology 111 (2017) 87–93

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Critical Reviews in Oncology/Hematology journal homepage: www.elsevier.com/locate/critrevonc

Immunotherapy for the treatment of multiple myeloma Sung-Hoon Jung a,b , Hyun-Ju Lee b , Manh-Cuong Vo b , Hyeoung-Joon Kim a , Je-Jung Lee a,b,∗ a b

Department of Hematology-Oncology, Chonnam National University Hwasun Hospital, Hwasun, Jeollanamdo, Republic of Korea Research Center for Cancer Immunotherapy, Chonnam National University Hwasun Hospital, Hwasun, Jeollanamdo, Republic of Korea

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Monoclonal antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 2.1. Elotuzumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 2.2. Daratumumab and other monoclonal antibodies targeting CD38 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 2.3. Immune checkpoint inhibitors and bispecific monoclonal antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Dendritic cell (DC) vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 3.1. Adoptive immunotherapy using genetically engineered T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 3.1.1. Chimeric antigen receptor T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 3.1.2. TCR-engineered T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

a r t i c l e

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Article history: Received 14 October 2016 Received in revised form 17 January 2017 Accepted 18 January 2017 Keywords: Myeloma Immunotherapy Monoclonal antibody Dendritic cell T cell

a b s t r a c t Immunotherapy has recently emerged as a promising treatment for multiple myeloma (MM). There are now several monoclonal antibodies that target specific surface antigens on myeloma cells or the checkpoints of immune and myeloma cells. Elotuzumab (targeting SLAMF7), daratumumab (targeting CD38), and pembrolizumab (targeting PD-1) have shown clinical activity in clinical studies with relapsed/refractory MM. Dendritic cell vaccination is a safe strategy that has shown some efficacy in a subset of myeloma patients and may become a crucial part of MM treatment when combined with immunomodulatory drugs or immune check-point blockade. Genetically engineered T cells, such as chimeric antigen receptor T cells or T cell receptor-engineered T cells, have also shown encouraging results in recent clinical studies of patients with MM. In this paper, we discuss recent progress in immunotherapy for the treatment of MM. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Multiple myeloma (MM) is an incurable B-cell malignancy characterized by the aberrant expansion of clonal malignant plasma cells into bone marrow that eventually causes renal failure, anemia, infection, and osteolytic bony lesions (Kyle and Rajkumar, 2004).

∗ Corresponding author at: Department of Hematology – Oncology, Chonnam National University Hwasun Hospital, 322 Seoyangro, Hwasun, Jeollanamdo, 519−763, Republic of Korea. E-mail address: [email protected] (J.-J. Lee). http://dx.doi.org/10.1016/j.critrevonc.2017.01.011 1040-8428/© 2017 Elsevier B.V. All rights reserved.

MM accounts for 1% of all cancers and more than 10% of all hematological malignancies in the United States (Siegel et al., 2015). The incidence of MM in Korea has rapidly increased in recent years (Lee et al., 2010). The prognosis for patients with MM has improved with the development of novel effective agents, and median survival has increased to approximately 6 years (Kumar et al., 2014). However, most patients with MM eventually relapse and develop resistance to their treatments. New therapies that increase the response and survival rates with minimal toxicity are needed. Immunotherapy has recently emerged as a promising treatment for many cancers. In MM, the efficacy of immunotherapy is based on the observation that allogenic stem cell transplantation is curative

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Table 1 Monoclonal antibodies and their targets in multiple myeloma. CD38

Daratumumab, Isatuximab, MOR202

SLAMF7 CD56 PD-1 CD138 CD40 CXCR4 FGFR3

Elotuzumab, Lorvotuzumab Pembrolizumab, Pidilizumab, Nivolumab Indatuximab Dacetuzumab Ulocuplumab MFGR1877S

for a subset of patients with MM due to the graft-versus-myeloma (GVM) effect (Tricot et al., 1996). In addition, the GVM effect is supported by disease response following donor lymphocyte infusions (Bellucci et al., 2004). However, allogenic stem cell transplantation does not have specific immune activity for myeloma cells and is associated with significant morbidity and mortality, including graft-versus-host disease. Therefore, investigators have focused on developing new tools to elicit myeloma-specific immune responses. An example of a new immunotherapeutic strategy is the development of a monoclonal antibody(mAb)-targeting surface antigen on myeloma cells (Table 1). Daratumumab, targeting CD38 and elotuzumab, targeting signaling lymphocyte activation molecule F7 (SLAMF7), have shown clinical activity in monotherapy or combination therapy with other agents in clinical studies. In addition, cellular immunotherapy using dendritic cell (DC) vaccination and adoptive immunotherapy with chimeric antigen receptor (CAR) T cells or T cell receptor (TCR)-engineered T cells are emerging as promising treatment strategies for MM. This review focuses on recent preclinical and clinical data from the dominant mAbs, DC vaccine, and genetically engineered T cell therapies for MM. 2. Monoclonal antibodies 2.1. Elotuzumab Elotuzumab is a first-in-class humanized IgG1 immunostimulatory mAb targeted to SLAMF7. It is also referred to as cell surface glycoprotein CD2 subset 1 (CS1), SLAMF7 is a glycoprotein expressed on myeloma cells and natural killer (NK) cells but not on normal tissue (Wang et al., 2016). It may play an important role in the interaction between myeloma cells and their adhesion to bone marrow stromal cells, which contributes to the survival and growth of myeloma cells. In addition, it plays an important role in NK cell activation (Cruz-Munoz et al., 2009). The mechanisms of the antitumor effects of elotuzumab include disrupting MM cell adhesion to bone marrow stromal cells, enhancing NK cell cytotoxicity, and mediating antibody-dependent cell-mediated cytotoxicity (ADCC), but not complement-mediated cytotoxicity (CDC). In a phase I study, elotuzumab was well tolerated in patients with advanced MM (Zonder et al., 2012). The most common adverse event was grade 1 or 2 infusion-related reaction, and 58.8% of patients experienced an infusion reaction during the first elotuzumab infusion. Although 26.5% of patients achieved disease stabilization, objective clinical responses were not seen with elotuzumab monotherapy. A clinical study of combination treatments with other approved drugs has been conducted, because elotuzumab showed encouraging anti-myeloma activity in preclinical studies when in combination with other agents (Tai et al., 2008; van Rhee et al., 2009). In a phase I study that evaluated the safety and efficacy of elotuzumab, lenalidomide, and dexamethasone in relapsed or refractory patients with MM, combination treatment resulted in a higher response rate (at least partial response, 82%) (Lonial et al., 2012), which compared favorably with the historical response rate of 60% using lenalidomide and dexamethasone

(Dimopoulos et al., 2007). These favorable results may be due to the synergistic activity of the two drugs: elotuzumab acts primarily through NK cell-mediated ADCC, and lenalidomide increases the number and anti-MM cytotoxic activity of NK cells. A phase II study also reported that the overall response rate was 84%, including 42% with a very good partial response (VGPR), and treatment was generally well tolerated (Richardson et al., 2015). In a randomized phase III study (ELOQUENT-2), patients treated with elotuzumab plus lenalidomide and dexamethasone had a higher response rate than patients treated with lenalidomie and dexamethasone (79% vs. 66%, P < 0.001), without a significant increase in adverse events. The median progression free survival (PFS) in the elotuzumab arm was 19.4 months, compared to 14.9 months in the lenalidomide/dexamethasone arm (Lonial et al., 2015). Bortezomib also enhanced the activity of elotuzumab in a preclinical study (van Rhee et al., 2009). In a phase I study, elotuzumab and bortezomib were well tolerated in patients with relapsed or refractory MM, with an overall response rate of 48% and median time to progression of 9.5 months (Jakubowiak et al., 2012). In a phase II study that evaluated the efficacy and safety of elotuzumab with bortezomib and dexamethasone compared to boretezomib and dexamethasone, median PFS was longer in the elotuzumab arm than the control arm (9.7 months vs. 6.9 months, P = 0.09). The overall response rate was also higher in the elotuzumab arm (66% vs. 63%) (Jakubowiak et al., 2016). 2.2. Daratumumab and other monoclonal antibodies targeting CD38 Daratumumab is a first-in-class human anti-CD38 IgG1k mAb. CD38 is a 45 kDa transmembrane glycoprotein that is highly expressed on malignant plasma cells, but is expressed at relatively low levels on normal lymphoid and myeloma cells (de Weers et al., 2011). Daratumumab binds CD38 on myeloma cells and induces cell death through several immune-mediated mechanisms, including CDC, ADCC, antibody-dependent cell phagocytosis (ADCP), induction of apoptosis, and modulation of CD38 enzyme activity (Overdijk et al., 2015). In addition, a recent study showed that daratumumab has immune-modulating effects through the reduction of CD38+ immunosuppressive cells and an increase in CD8+ cytotoxic T cells and CD4+ helper T cells in patients with relapsed or refractory MM (Krejcik et al., 2016). A previous phase I/II study (GEN501) utilized a 3 + 3 doseescalation design with daratumumab administration, that ranged from 0.005 to 24 mg per kg of body weight (Lokhorst et al., 2015). The maximum tolerated dose was not reached with the use of doses up to 24 mg/kg. In patients treated with a dose of 16 mg/kg, the overall response rate was 36%. The median PFS was 5.6 months, and the overall survival (OS) rate at 12 months was 77%. The SIRIUS study reported similar results (Lonial et al., 2016). Overall responses were noted in 29.2% of patients treated with 16 mg/kg. Furthermore, at least a partial response (PR) was achieved in 21% of patients who were refractory to four drugs (bortezomib, lenalidomide, pomalidomide, and carfilzomib). These data suggest that resistance to previous therapy did not affect the activity of daratumumab. The median PFS was 3.7 months, and the 12-month OS was 64.8%. Daratumumab treatment was generally safe, and most of the common non-hematological adverse events were infusion-related reactions, such as fever, cough, nausea, dizziness, and bronchospasm. Most infusion-related reactions occurred in the first infusion, and the infusion rate may be associated with the development of infusionrelated reactions. In an in vitro study, combinations of daratumumab and lenalidomide significantly increased lysis of MM cells, mainly due to the potent capacity of lenalidomide to activate ADCC effector cells (van der Veer et al., 2011b). In addition, bortezomib enhanced the ther-

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apeutic effects of daratumumab by sensitizing myeloma cells for antibody-mediated lysis (van der Veer et al., 2011a). In a phase I/II study, a combination of daratumumab, lenalidomide, and dexamethasone resulted in an improved response rate in relapsed or refractory patients with MM: with an overall response rate of 81% with 25% stringent CR, 9% CR, and 28% VGPR. The PFS and OS rate at 18 months were 72% and 90%, respectively (Plesner et al., 2016). Based on these results, a randomized phase III study (POLLUX) that used daratumumab, lenalidomide, and dexamethasone versus lenalidomide and dexamethasone alone are ongoing in relapsed or refractory patients with MM. Recently, a randomized phase III study (CASTOR trial) was conducted in patients with relapsed or refractory MM (Palumbo et al., 2016). Patients treated with daratumumab, bortezomib, and dexamethasone had a significantly higher response rate than those treated with bortezomib and dexamethasone alone (overall response rate 82.9% vs. 63.2%, P < 0.001; ≥VGPR 59.2% vs. 29.1%, P < 0.001; ≥CR 19.2% vs. 9.0%, P = 0.001), longer PFS, and higher rates of thrombocytopenia (45.3% vs. 32.9%) and neutropenia (12.8% vs. 4.2%). Isatuximab (SAR650984) is another potent mAb against CD38. Besides effector-mediated ADCC, CDC, and ADCP, it has direct cytotoxicity in the absence of cross-linking agents or immune effector cells (Jiang et al., 2016). In addition, it induces more potent inhibition of CD38 enzymatic activity compared to other CD38 antibodies (Bueren et al., 2014). In a phase II study, the overall response rate of isatuximab monotherapy was 24% in 74 patients with relapsed or refractory MM who received dose levels ≥10 mg/kg and the median duration of the response was 6.6 months (Richter et al., 2016). Infusion-related reactions occurred in 49% of patients, mostly grade ≤ 2. A combination of isatuximab, lenalidomide, and dexamethasone was well tolerated and showed an overall response rate of 57% with a median duration of response of 7.6 months (Vij R. et al., 2016). Clinical studies of isatuximab with carfilzomib for relapsed or refractory MM (NCT02332850), and isatuximab, bortezomib, cyclophosphamide, and dexamethasone for newly diagnosed MM (NCT02513186) are ongoing. MOR202 is a humanized IgG1-mAb against CD38, and preliminary data on its safety and efficacy were reported in a phase I/IIa study in relapsed or refractory multiple myeloma. MOR202 was well tolerated and infusion-related reactions occurred in 14% of the patients when MOR202 was combined with dexamethasone. A high percentage of CR has been observed in patients receiving MOR202 in combination with pomalidomide and dexamethasone (Raab et al., 2016). 2.3. Immune checkpoint inhibitors and bispecific monoclonal antibodies Immune-check point inhibitors targeting the PD-1/PD-L1 axis have recently emerged as promising agents that control antitumor immune responses. The binding between PD-L1 expressing tumor cells and PD-1 on T lymphocytes results in the inhibition of T cell proliferation, cytokine secretion, and an increase in regulatory T cells (Treg), which collectively result in immune tolerance (Francisco et al., 2009). Antibodies to PD-1 and PD-L1 prevent the activation of the immune inhibitory pathway and have shown an excellent clinical activity in various types of cancers. In MM, clinical trials targeting the PD-1/PD-L1 axis are being conducted. In a phase I trial, combinations of pembrolizumab, lenalidomide, and dexamethasone in relapsed or refractory MM showed an acceptable toxicity profile and promising activity, with an overall response rate of 76% with 24% VGPR and 53% PR (Miguel et al., 2015). In addition, the combination of pembrolizumab, pomalidomide, and dexamethasone had a 50% objective response including 13.6% near CR, 9% VGPR, and 27.3% PR, in heavily treated MM (Badros et al., 2015). Nivolumab is another PD-1-bolcking antibody that shows favorable

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safety with modest clinical activity as a single agent, with 4% CR and 63% stable disease (SD) in 27 patients with relapsed or refractory MM (Lesokhin et al., 2016). A bispecific antibody therapeutic strategy has emerged as a promising therapeutic approach for acute lymphoblastic leukemia and some types of lymphoma (Huehls et al., 2015). Bispecific T cell Engager (BiTE) antibody for myeloma treatment has also been actively investigated in vitro and in vivo (Bianchi et al., 2015). BiTE antibody is composed of two single-chain variable fragments (scFvs) connected by a flexible linker. One scFv binds to a T cell-specific antigen (typically CD3), whereas the other binds to a tumor-specific antigen. These structure and specificity allow BiTEs to juxtapose T cells and tumor cells physically and promotes the formation of immunological synapses by the simultaneous binding of multiple BiTEs, leading to T cell activation, cytokine production and cytotoxicity of the tumor cells. The most advanced clinical trial of a BiTE involves blinatumomab (MT103; CD3-CD19 BiTE), which targets CD19+ malignancies (Topp et al., 2015). It was granted FDA approval with a 43% CR rate in relapsed or refractory B cell-precursor acute lymphoblastic leukemia. CD3-CD38 and CD3CD138 BiTEs are under preclinical development for MM (Chen et al., 2016; Zou et al., 2015)., phase I dose escalation study of BCMA BiTE antibody (BI836909) is currently ongoing (NCT02514239).

3. Dendritic cell (DC) vaccines Dendritics cells (DCs) are the most important antigenpresenting cells that recognize, process, and present antigens on the cell surface to naïve T cells and modulate tumor-specific immunity. However, circulating DCs in patients with MM have quantitative and functional deficiencies that contribute to tumor-associated immune tolerance (Rosenblatt and Avigan, 2008). The maturation of DCs is also inhibited by factors released from myeloma cells such as vascular endothelial growth factor, tumor growth factor-␤, and interleukin (IL)-10 (Brown et al., 2001; Ratta et al., 2002). Restoration of dysfunctional DCs is crucial for generating an effective DC vaccine. In a previous study, we demonstrated that DCs generated ex vivo from patients with myeloma had a functionally active phenotype and could generate potent myeloma-specific cytotoxic T lymphocytes (CTLs) (Lee et al., 2007; Yang et al., 2009). The initial clinical study with DCs generated ex vivo was performed in patients with refractory myeloma using an idiotype (Id) protein as tumor antigen. Although Id-pulsed DC vaccination showed immunological response in a subset of patients, clinical response was observed in few patients(Liso et al., 2000; Wen et al., 1998). The reasons for these insufficient results may be that Id proteins are a weak antigen and Id-pulsed DCs target a single tumor antigen. An alternative to enhance the efficacy of DCvaccination is to use antigens derived from whole myeloma cells taken from patients with MM. DCs loaded with the entire myeloma cell-derived antigen have the advantage of allowing the presentation of multiple epitopes to MHC on DCs, thus inducing polyclonal T cell responses from many potentially unknown tumor-associated antigens (Nguyen-Pham et al., 2012). In a preclinical study, DCs pulsed with an optimal concentration of myeloma cell lysates could generate potent myeloma-specific CTLs, inducing strong cytotoxic activity against autologous myeloma cells (Lee et al., 2007). Rosenblatt et al. (Rosenblatt et al., 2011b) conducted a phase I clinical study with DC/tumor fusion cells in patients with relapsed MM that resulted in disease stabilization in 66% of patients. Vaccination with DC/myeloma cell fusions following autologous stem cell transplantation for patients with MM induced the marked expansion of myeloma-specific T cells and the cytoreduction of minimal residual disease (Rosenblatt et al., 2013). A phase II trial is currently ongoing that uses lenalidomide maintenance with

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Fig. 1. Preparation of DC vaccines for clinical use. Ex-vivo DCs can be generated from several sources, matured with various stimuli and loaded with various tumor-associated antigens using a variety of techniques. Numerous variables, such as cell dose and frequency and route of administration, also need to be optimized for an effective DC vaccination. Multiple modality targeting with adjuvants, immunomodulatory drugs and therapeutic agents is necessary to enhance the efficacy of DC-based cancer immunotherapy.

or without DC/myeloma cell fusion vaccination during the posttransplant period in MM patients. MM-specific antigen-pulsed DCs are another attractive option that can be generalized to DC-based immunotherapy for MM patients. A variety of tumor-associated antigens has been identified in MM patients, and some have been tested as peptide vaccines in vitro and in vivo. DCs pulsed with multiple peptides such as XBP1-US184-192 (YISPWILAV), heteroclitic XBP1-SP367-375 (YLFPQLISV), native CD138260-268 (GLVGLIFAV), and native CS1239-247 (SLFVLGLFL) induce antigen-specific CTLs targeting myeloma cells in vitro (Bae et al., 2012). In a phase I study that used a tumor-associated antigen-mRNA-loaded DC vaccination in 12 MM patients with minimal residual disease after autologous SCT, all patients showed strong anti-KLH T cell responses, and MAGE3specific CD4+ and CD8+ T cells, as well as CD3+ T cells reactive against BCMA and survivin, were detected in one patient (Hobo et al., 2013). These data suggest that DCs pulsed with myelomaspecific antigens are a promising treatment for MM. Recently, our group developed potent DCs loaded with dying myeloma cells that induced the increased migration capacity of DCs, the strong Th1 polarization of naïve T cells, and potent myeloma-specific CTL generation (Nguyen-Pham et al., 2011; Yang et al., 2010; Yang et al., 2011). In a phase I/IIa study for patients with relapsed or refractory MM, vaccination with potent DCs loaded with dying myeloma cells was well tolerated and showed diseasestabilizing activity in 66.7% of patients, with a 77.8% immunological response (Jung et al., 2017). Although immunotherapy with the DC vaccine has developed gradually, the clinical efficacy of the DC-based vaccine must be enhanced using new treatment strategies. Lenalidomide is a potent immunomodulatory drug that enhances T cell expansion with Th1 polarization by inhibiting of Treg development and PD-1 expression, activating NK cells and T cells, suppressing inhibitory factors, and enhancing tumor–specific immune responses (Neuber et al., 2011). These activities have affected the response to DC-based therapy. Previously, we reported that a combination of lenalidomide

and DC vaccine synergistically enhanced anti-tumor immunity by inhibiting immune suppressor cells and recovering effector cells in a mouse myeloma model (Nguyen-Pham et al., 2015). In addition, recent data suggest that an inhibitory receptor blockade by a PD-1 inhibitor stimulated the proliferation and cytokine secretion of exhausted CD8+ T cells in vitro (Rosenblatt et al., 2011a). A combination treatment with these agents that can modulate the immunosuppressive microenvironment may be a more realistic approach to enhancing antitumor effects than monotherapy with DC-based vaccines in the future (Fig. 1). 3.1. Adoptive immunotherapy using genetically engineered T cells 3.1.1. Chimeric antigen receptor T cells Chimeric antigen receptors (CARs) are synthetic, engineered receptors that can target surface molecules in their native conformation. Unlike TCRs, CARs engage molecular structures and target surface antigens in a major histocompatibility complex (MHC)-independent fashion. In addition, unlike TCRs, CARs typically have a much higher and broader range of affinities to targets. First-generation CARs were constructed with a single-chain variable fragment derived from an antibody and intracellular CD3zeta signaling domain without any co-stimulation. While these firstgeneration CAR T cells were able to target an antigen specifically, they had very modest clinical activity and poor in vivo persistence. To overcome these limitations, co-stimulatory molecules such as CD27, CD28, CD134 (OX40), or CD137 (4-1BB) have been incorporated into second-generation CAR T cells to provide different effector functions such as proliferation and cytokine production. Third-generation CAR T cells contain an additional co-stimulatory domain, such as CD28 plus 4-1BB or CD28 plus OX40 (Fig. 2A) (Maus et al., 2014). In a recent study, 90% of relapsed or refractory patients with acute lymphoblastic leukemia (ALL) who received CAR T cells targeting CD19 achieved complete remission (Maude et al., 2014). In

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Fig. 2. Anatomy of CAR and TCR constructs. (A) CARs target surface antigens in an MHC-independent fashion and consist of an ectodomain (scFv: a single chain-variable fragment derived from an antibody), hinge domain, transmembrane domain, and endodomain (intracellular CD3zeta signaling domain with co-stimulation 1 and/or 2). Current clinical trials are testing second- and third-generation CARs. (B) TCRs consist of ␣- and ␤-chains derived from tumor-specific T cells. When the TCR encounters tumor antigenic peptide fragments presented in an MHC-dependent fashion, it leads to a cascade of intracellular signaling that results in the release of cytokines and cytotoxic molecules from T cells (abbreviations: VH, heavy chain of variable region; VL , light chain of variable region; V, variable region; C, constant region).

MM, B cell maturation antigen (BCMA), CS1, CD38, CD138, and CD56 are considered target antigens for CAR T cell therapy. BCMA is a transmembrane protein that regulates B cell maturation and differentiation into plasma cells, and is one of the major target antigens since it is mainly expressed on plasmablast and plasma cells (Tai et al., 2006). Treatment with BCMA-targeted CAR T cells resulted in a 90–100% clearance of bone marrow plasma cells (Ali et al., 2015). Recently, 12 patients with MM received CAR-BCMA T cells in a dose-escalation clinical trial, and the duration of myelomaspecific response was documented (Ali et al., 2016). Among the six patients treated with the two lowest dose levels (0.3 × 106 and 1 × 106 CAR T cells/kg body weight, respectively), limited antimyeloma activity (1 PR and 5 SD) and mild toxicity was observed. At the third dose level (3 × 106 CAR T cells/kg body weight), one of three patients showed VGPR and two showed SD. Furthermore, one patient at the fourth dose level (9 × 106 CAR T cells/kg body weight) obtained stringent CR that lasted for 17 weeks before relapse, and the other patient currently shows VGPR, demonstrating the powerful anti-myeloma activity of CAR-BCMA T cells in patients with MM. In addition, Garfall et al. (Garfall et al., 2015) reported that a patient treated with CAR T cells against CD19 showed CR with no evidence of progression at 12 months after treatment. This result is interesting because CD19 is lost during the maturation of B cells to plasma cells and is detected in less than 5% of MM cells (Mateo et al., 2008). CS1 is considered a promising target for CAR T cells and clinical trials are expected to begin soon. It is expressed on some activated T cells. It can lead to fratricide of CAR T cells but this can be overcome with gene-editing techniques to knock-out the CS1 gene in CAR-transduced cells. In addition, early phase clinical trials with CAR-T cells targeting the ␬ light chain, Lewis Y-antigen, and NKG2D ligands are ongoing in various types of hematologic malignancy, including a few patients with MM. It is unclear what is the optimal target antigen for CAR T-cell therapy, and whether simultaneous targeting of multiple antigens will be needed to eliminate the myeloma cells. There are still some barriers in terms of safety, such as “on-target, off-tumor” toxicity, cytokine release syndrome, and neurological toxicities (Dai et al., 2016). With regard to the risk of “on-target, off-tumor” toxicity, B cell aplasia and GVHD were

on-target results of successful CD19-specific CAR T cell therapy. Therefore, very tumor-specific antigen molecules must be chosen to target and refine the affinity and specificity of CAR, and the conditioning regimens used prior to cell infusion to avoid untoward outcomes are important. Cytokine release syndrome is frequently accompanied by high fever, hypotension, and hypoxia, potentially resulting in organ failure, and is related to the production of several pro-inflammatory cytokines, including IL-6, TNF␣, and IFN␥, secondary to CAR T cell activation. Cytokine-blocking drugs such as the IL-6 receptor antagonist tocilizumab and steroids have been used to control cytokine release syndrome without compromising T cell efficacy. A handful of ALL patients treated with anti-CD19 CAR T cells in some clinical trials have shown neurological toxicities including delirium, dysphasia, akinetic mutism, and seizures. The mechanism of these symptoms is still unclear and warrants careful and greater investigation. 3.1.2. TCR-engineered T cells TCR genes, consisting of ␣- and ␤-chains, can be derived from tumor-specific T cells, which can naturally occur in humans, or from the immunization of HLA-transgenic mice. The ␣- and ␤-chains associate with the ␥-, ␦-, ␧-, and ␨-chains of the CD3 complex. When the TCR encounters tumor antigenic peptide fragments presented on the MHC of the tumor cells, immunoreceptor tyrosine-base activation motifs (ITAMs) are phosphorylated, leading to a cascade of intracellular signaling that results in the release of cytokines and cytotoxic molecules from T cells (Fig. 2B). An advantage of using TCR genes to endow specificity is that the TAA can be derived from the entire protein composition of tumor cells, including intracellular proteins. In addition, there is the potential for truly tumor-specific responses with the identification of specific mutant proteins that are restricted to tumor cells. However, each TCR gene can be used only in a proportion of patients, owing to the MHC-restricted nature of TCR function. Therefore, to broaden the application of gene-engineered T cells, genes encoding CARs, which operate in a non-MHC-restricted manner, have been generated (Kershaw et al., 2013; Morris and Stauss, 2016). NY-ESO-1- and LAGE-1 TCR-engineered T cells have been employed in a phase I/II clinical trial for the treatment of myeloma

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(Rapoport et al., 2015). Twenty patients with antigen-positive MM received an average 2.4 × 109 engineered T cells after autologous stem cell transplantation. Infusions were well-tolerated without clinically apparent cytokine release syndrome, despite high levels of IL-6. Clinical response was remarkable, with 70% having near CR/CR, 10% VGPR, and 10% PR. The median progression-free survival was 19.1 months. Disease progression was correlated with loss of persistence of the TCR-engineered T cells or with loss of NYESO-1 expression in the myeloma cells, suggesting that immune editing allowed myeloma cells to escape T cell attack. 4. Conclusions The improvement of survival outcomes in patients with MM has primarily resulted from high-dose chemotherapy/autologous stem cell transplantation and the introduction of novel agents such as thalidomide, bortezomib, and lenalidomide. However, further treatment strategies are still required to overcome resistance stemming from previous treatments, and to cure MM. The development of cancer immunotherapy using mAbs, DC vaccines, and genetically engineered T cells for MM may represent a new era for the treatment of MM. The results from preclinical and clinical trials have been striking, and results of ongoing clinical trials are anticipated. These new techniques may allow us to develop a cure for MM in the near future. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgments This study was supported financially by the Leading Foreign Research Institute Recruitment Program (2011-0030034) and 2015R1D1A1A09057809 through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (MEST), and by the Korea Health Technology R&D Project (HI14C1898) through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea. References Ali, S.A., Shi, V., Wang, M., Stroncek, D., Maric, I., Brudno, J.N., et al., 2015. Remissions of multiple myeloma during a first-in-humans clinical trial of t cells expressing an anti-B-Cell maturation antigen chimeric antigen receptor. Blood 126, LBA-1. Ali, S.A., Shi, V., Maric, I., Wang, M., Stroncek, D.F., Rose, J.J., et al., 2016. T cells expressing an anti-B-cell-maturation-antigen chimeric antigen receptor cause remissions of multiple myeloma. Blood 128 (13), 1688–1700. Badros, A.Z., Kocoglu, M.H., Ning Ma Rapoport, A.P., Lederer, E., Philip, S., et al., 2015. A phase II study of anti PD-1 antibody pembrolizumab, pomalidomide and dexamethasone in patients with relapsed/refractory multiple myeloma (RRMM). Blood 126, 506. Bae, J., Smith, R., Daley, J., Mimura, N., Tai, Y.T., Anderson, K.C., et al., 2012. Myeloma-specific multiple peptides able to generate cytotoxic T lymphocytes: a potential therapeutic application in multiple myeloma and other plasma cell disorders. Clin. Cancer Res. 18 (17), 4850–4860. Bellucci, R., Wu, C.J., Chiaretti, S., Weller, E., Davies, F.E., Alyea, E.P., et al., 2004. Complete response to donor lymphocyte infusion in multiple myeloma is associated with antibody responses to highly expressed antigens. Blood 103 (2), 656–663. Bianchi, G., Richardson, P.G., Anderson, K.C., 2015. Promising therapies in multiple myeloma. Blood 126 (3), 300–310. Brown, R.D., Pope, B., Murray, A., Esdale, W., Sze, D.M., Gibson, J., et al., 2001. Dendritic cells from patients with myeloma are numerically normal but functionally defective as they fail to up-regulate CD80 (B7-1) expression after huCD40LT stimulation because of inhibition by transforming growth factor-beta1 and interleukin-10. Blood 98 (10), 2992–2998. Bueren, J.L.v., Jakobs, D., Kaldenhoven, N., Roza, M., Hiddingh, S., Meesters, J., et al., 2014. Direct in vitro comparison of daratumumab with surrogate analogs of CD38 antibodies MOR03087, SAR650984 and ab79. Blood 124, 3474.

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