**5. Combination therapy: New concept to enhance efficacy of DC vaccines**

Many factors contribute to the limited clinical efficacy of DC vaccines. The tumor microenvir‐ onment contains different kinds of inhibitory cells, such as Tregs and MDSC, and inhibitory molecules, such as IL-10, IL-6, TGF-β, and VEGF, all of which prevent the activation of effector T cells in response to DC responses [16-21, 23, 78, 79]. Although DC vaccines showed effective antitumor effect in experimental *ex vivo* systems, they didn't effectively induce strong immune responses that were enough to kill tumors *in vivo*. Therefore, strategies to improve the efficacy of DC vaccines are to overcome the immune tolerance/suppression induced by these cells, which are involved in the use of a combination of DC vaccine with either stimulatory cytokines or the targeting elimination of inhibitory cells and molecules in tumor microenvironment.

#### **5.1. DC vaccine and cytokine combination**

cell polarization that was dependent on the activation of IL-12p70 and independent of TLR4 [71, 72]. The potential of natural products to enhance DC maturation and activation has

Another important consideration to improve the efficacy of DC vaccination in patients with MM is an effective tumor antigen, instead of using idiotype proteins with a weak antigenicity. The use of whole tumor cells, instead of single antigen, may help to enhance antitumor effects to target multiple tumor variants. It is necessary to use purified, optimized myeloma cells, if possible, as a source of tumor antigens for loading onto DCs to generate potent myelomaspecific CTLs [35]. However, it is not only impractical to obtain sufficient amounts of purified autologous myeloma cells for tumor antigens in the clinical setting from patients with MM and it is also unsuitable for those with a lower tumor burden status. As an alternative source of tumor-relevant antigens, allogeneic tumor cells or established cancer cell lines have been used to overcome the limitation in various tumors [37, 38]. DCs loaded with tumor antigens derived from allogeneic myeloma cells could generate myeloma-specific CTLs against autologous myeloma cells in patients with MM [37, 39]. The success of using an allogeneic myeloma cell line as tumor antigens led to the possibility that allogeneic myeloma cells could also be used as a viable source of tumor antigens in the context of appropriate major MHC alleles to autologous CTLs. In addition, autologous DCs loaded with dying myeloma cells of allogeneic matched monoclonal immunoglobulin subtype showed to generate potent myelo‐ ma-specific CTLs against autologous myeloma cells in MM patients [38] These findings suggested that allogeneic myeloma cell lines and allogeneic matched monoclonal immuno‐ globulin subtype of myeloma were effective tumor antigens capable of inducing functional

Improved understanding of which specific anticancer agents lead to immunogenic cell death and whether these process can enhance antitumor immunity may facilitate the mechanism how chemotherapy and immunotherapy combination can induce immune responses against cancer. Recently, we have worked to develop strategies that recover dysfunction of DCs caused from loading tumor antigens through treatment of myeloma cells with a combination of the selective JAK/STAT3 inhibitor, JSI-124, and a kind of proteasome inhibitor, bortezomib. We observed that production of inhibitory cytokines, such as IL-10, IL-23, and especially IL-6, which induces DC dysfunction in MM patients, was down-regulated in DCs loaded with dying myeloma tumor cells that induced by these agents. Furthermore, phospho-STAT3 was also down-regulated in the DCs. These DCs displayed a superior ability to induce myeloma-specific responses of CTLs. More recently, we are investigating whether chaetocin could be used to induce dying tumor cells for loading onto DCs to enhance myeloma-specific antitumor responses. We show that anti-myeloma drug-induced dying tumor cells can be used as the source of myeloma antigens to loading onto DCs that could elicit potent anti-myeloma activity of CTLs due to the expression of HSP and cancer testis antigens as a mechanism of immuno‐

important implications for the use of DCs as a cancer vaccine.

184 Multiple Myeloma - A Quick Reflection on the Fast Progress

CTLs against patients' own myeloma cells.

genic death of human MM cells.

**4.3. New sources of myeloma-associated antigens for DC vaccines**

Cytokines, such as GM-CSF or IL-2, known to enhance cell-mediated immune responses may be administered as adjuvants with the vaccines aiming to create an environment where specific immune responses are readily induced [80, 81]. To enhance the efficacy of DC vaccination, Idpulsed DCs were combined with GM-CSF [80, 82-84], with immunogenic carrier molecules such as KLH [27, 28, 31-33, 82, 85], or cytokine IL-2 [80, 83] to improve the effectiveness of these DC vaccines in patients with MM. Recently, a phase I study was performed in patients with MM using autologous DCs/tumor cells fusion in combination with GM-CSF administration at the day of DC vaccination [86]. The expansion of circulating CD4+ and CD8+ T cells reactive with autologous myeloma cells were detected in 11 of 15 evaluable patients. A majority of patients with advanced disease demonstrated disease stabilization. In a murine myeloma model, mice were vaccinated with DC-plasmacytoma cell fusions and demonstrated that administration of IL-12 with the vaccine resulted in potentiation of *in vivo* T cell proliferation and cytotoxicity and eradication of established disease [87]. Therefore, the combination of DC vaccine with stimulatory cytokines is a feasible approach to provide a new source of DC-based vaccines for the development of immunotherapy against MM.

a treatment combining the lenalidomide with DC vaccination markedly improved antitumor effect by inhibiting immunosuppressor cells, recovering effector cells, and inducing superior polarization of the Th1/Th2 balance in favor of the Th1 response. This immunomodulatory effect may be a crucial component of the enhancer-like properties of lenalidomide in the context

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http://dx.doi.org/10.5772/54100

187

**5.3. Chemotherapeutic agent can induce "immunogenic myeloma-cell death" to trigger**

Most of chemotherapeutic agents kill tumor cells by the induction of apoptosis. Previously, chemotherapy and immunotherapy have usually been regarded as unrelated therapy in the treatment of cancers because chemotherapy-induced apoptotic cell death has long been considered as non-immunogenic or inducing immune tolerance. Recently, apoptotic cell death when coupled with inflammatory signals, such as HSPs, is clearly known to induce the activation of DCs and triggers the immune response [100]. Some chemotherapeutic agents could induce a type of tumor cell death that activates efficient antitumor immunity, so it is called "immunogenic tumor-cell death". Immunogenic tumor-cell death expresses danger signals on the tumor cell surface or secretes immunostimulatory factors, such as HSPs, calreticulin, high mobility group box 1 protein (HMGB1), and ATP, into the tumor microen‐ vironment, thereby promoting DC maturation and stimulating a powerful T cell immune

Cyclophosphamide is well known as a potent cytotoxic and lymphoablative drug in conven‐ tional and high dosages. However, more recent work highlighted as an immunostimulatory and/or antiangiogenic agent at low dosages, openning up novel indication in the field of cancer immunotherapy. In recent reports, cyclophosphamide administration in tumor-bearing mice induced pre-apoptotic surface translocation of calreticulin on tumor cells [101], which serves as an "eat-me" signal for phagocytes [102] and the release of high-mobility group box1 (HMGB1) protein in the extracellular milieu [101], which constitutes a "danger signal" triggering activation of the DC processing machinery [103]. These events are prerequisites for adequate engulfment of tumor apoptotic material and optimal CD8+ T cell cross-priming by

HSPs are intracellular chaperones for many proteins, but they can also be expressed on the cell surface or even be released under stress conditions [104, 105]. HSP acts as an adjuvant in initiating the activation of DCs or as protein vehicle to facilitate the presentation of antigen peptides to T cells. Spisek et al. [106] reported that uptake of myeloma cells by DCs after tumor cell death induced by bortezomib leads to the induction of antitumor immunity and enhances DC-mediated tumor immune response, indicating the probability mechanism due to the expression of HSP90 on the surface of dying cells, thereby facilitating the activation of DCs in response to dying tumor cells. Our study also found that HSPs released from dying tumor cells, which were induced by a combination of the selective JAK/STAT3 inhibitor JSI-124 and proteasome inhibitor bortezomib, act on tumor cells to recover DC dysfunction and to induce cytokine and chemokine production from DCs, resulting in generation of potent myeloma-

of antitumor immunity against MM.

response [88].

DCs [102, 103].

specific CTL response against myeloma cells.

**activation of DCs and to enhance cross-presentation of DCs**

#### **5.2. DC vaccine and chemotherapy combination**

Chemotherapy can help to reverse the immunosuppression caused by cancers and also further enhance the capacity of DCs to trigger antitumor immunity [88]. Accumulating evidence indicates that conventional chemotherapy as well as radiotherapy selectively eliminates immunosuppressive cells, triggers the activation of DCs, and enhances antigen cross-presen‐ tation. Furthermore, specific anticancer agents lead to immunogenic cell death of tumor cells and these processes can enhance antitumor immunity.

Recent studies have shown that chemotherapeutic agents increase the efficacy of active or adoptive antitumor immunotherapies through beneficial immunomodulatory effects [89, 90]. Cyclophosphamide eliminates the activities of tumor-induced suppressor T cells in tumorbearing hosts [90] and induces the production of immunostimulatory cytokines, such as type I IFN [91]. In addition, low-dose cyclophosphamide has been shown to down-regulate suppressor T cells and to decrease the production of TGF-β and IL-10 while inducing a Th2/Th1 shift in the cytokine profile [92-94]. Low-dose cyclophosphamide may enhance the antitumor efficacy of DC vaccines by increasing the proportion of IFN-γ secreting lymphocytes and suppressing the proportion of CD4+ CD25+ FoxP3+ Tregs in tumor-bearing mice [95]. The result of a clinical trial using allogeneic DC vaccines combined with low-dose cyclophospha‐ mide has revealed that the combination therapy could induce stronger antitumor responses compared to the DC vaccine alone [96]. Recently, we demonstrated that a single administration of low-dose cyclophosphamide before the first DC vaccination showed to augment antitumor effects of DC vaccines to completely eradicate the tumor and to prolong the survival of vaccinated mice [64].

Lenalidomide is a thalidomide analog that has more potent anti-myeloma effects and less adverse effects [97]. Lenalidomide can induce apoptosis of myeloma cells, inhibit the produc‐ tion of cytokines (IL-6, VEGF, and TNF-α) in bone marrow of myeloma patients, and stimulate T cell and NK cell proliferation, cytotoxicity, and cytokine (IL-2, IFN-γ) production [97]. In addition, lenalidomide can inhibit the frequency and function of Tregs, resulting in inhibition of Treg expansion and FoxP3 expression in cancer patients patients [98]. Interestingly, this drug can also induce the activation of APC function, resulting in upregulation of CD40, CD80, and CD86 in chronic lymphocytic leukemia [99]. Therefore, lenalidomide can be used as an immunomodulatory drug in order to enhance immune responses against cancer. Our *in vitro* study showed that lenalidomide enhanced the maturation and function of DCs in the presence of LPS, resulting in synergistic stimulation of DCs to increase phenotype expression, IL-12p70 production, T cell stimulation capacities, and CTL activities against myeloma cells, and to suppress the generation of Tregs. Moreover, our *in vivo* mouse myeloma model showed that a treatment combining the lenalidomide with DC vaccination markedly improved antitumor effect by inhibiting immunosuppressor cells, recovering effector cells, and inducing superior polarization of the Th1/Th2 balance in favor of the Th1 response. This immunomodulatory effect may be a crucial component of the enhancer-like properties of lenalidomide in the context of antitumor immunity against MM.

patients with advanced disease demonstrated disease stabilization. In a murine myeloma model, mice were vaccinated with DC-plasmacytoma cell fusions and demonstrated that administration of IL-12 with the vaccine resulted in potentiation of *in vivo* T cell proliferation and cytotoxicity and eradication of established disease [87]. Therefore, the combination of DC vaccine with stimulatory cytokines is a feasible approach to provide a new source of DC-based

Chemotherapy can help to reverse the immunosuppression caused by cancers and also further enhance the capacity of DCs to trigger antitumor immunity [88]. Accumulating evidence indicates that conventional chemotherapy as well as radiotherapy selectively eliminates immunosuppressive cells, triggers the activation of DCs, and enhances antigen cross-presen‐ tation. Furthermore, specific anticancer agents lead to immunogenic cell death of tumor cells

Recent studies have shown that chemotherapeutic agents increase the efficacy of active or adoptive antitumor immunotherapies through beneficial immunomodulatory effects [89, 90]. Cyclophosphamide eliminates the activities of tumor-induced suppressor T cells in tumorbearing hosts [90] and induces the production of immunostimulatory cytokines, such as type I IFN [91]. In addition, low-dose cyclophosphamide has been shown to down-regulate suppressor T cells and to decrease the production of TGF-β and IL-10 while inducing a Th2/Th1 shift in the cytokine profile [92-94]. Low-dose cyclophosphamide may enhance the antitumor efficacy of DC vaccines by increasing the proportion of IFN-γ secreting lymphocytes

CD25+

result of a clinical trial using allogeneic DC vaccines combined with low-dose cyclophospha‐ mide has revealed that the combination therapy could induce stronger antitumor responses compared to the DC vaccine alone [96]. Recently, we demonstrated that a single administration of low-dose cyclophosphamide before the first DC vaccination showed to augment antitumor effects of DC vaccines to completely eradicate the tumor and to prolong the survival of

Lenalidomide is a thalidomide analog that has more potent anti-myeloma effects and less adverse effects [97]. Lenalidomide can induce apoptosis of myeloma cells, inhibit the produc‐ tion of cytokines (IL-6, VEGF, and TNF-α) in bone marrow of myeloma patients, and stimulate T cell and NK cell proliferation, cytotoxicity, and cytokine (IL-2, IFN-γ) production [97]. In addition, lenalidomide can inhibit the frequency and function of Tregs, resulting in inhibition of Treg expansion and FoxP3 expression in cancer patients patients [98]. Interestingly, this drug can also induce the activation of APC function, resulting in upregulation of CD40, CD80, and CD86 in chronic lymphocytic leukemia [99]. Therefore, lenalidomide can be used as an immunomodulatory drug in order to enhance immune responses against cancer. Our *in vitro* study showed that lenalidomide enhanced the maturation and function of DCs in the presence of LPS, resulting in synergistic stimulation of DCs to increase phenotype expression, IL-12p70 production, T cell stimulation capacities, and CTL activities against myeloma cells, and to suppress the generation of Tregs. Moreover, our *in vivo* mouse myeloma model showed that

FoxP3+

Tregs in tumor-bearing mice [95]. The

vaccines for the development of immunotherapy against MM.

**5.2. DC vaccine and chemotherapy combination**

186 Multiple Myeloma - A Quick Reflection on the Fast Progress

and these processes can enhance antitumor immunity.

and suppressing the proportion of CD4+

vaccinated mice [64].

#### **5.3. Chemotherapeutic agent can induce "immunogenic myeloma-cell death" to trigger activation of DCs and to enhance cross-presentation of DCs**

Most of chemotherapeutic agents kill tumor cells by the induction of apoptosis. Previously, chemotherapy and immunotherapy have usually been regarded as unrelated therapy in the treatment of cancers because chemotherapy-induced apoptotic cell death has long been considered as non-immunogenic or inducing immune tolerance. Recently, apoptotic cell death when coupled with inflammatory signals, such as HSPs, is clearly known to induce the activation of DCs and triggers the immune response [100]. Some chemotherapeutic agents could induce a type of tumor cell death that activates efficient antitumor immunity, so it is called "immunogenic tumor-cell death". Immunogenic tumor-cell death expresses danger signals on the tumor cell surface or secretes immunostimulatory factors, such as HSPs, calreticulin, high mobility group box 1 protein (HMGB1), and ATP, into the tumor microen‐ vironment, thereby promoting DC maturation and stimulating a powerful T cell immune response [88].

Cyclophosphamide is well known as a potent cytotoxic and lymphoablative drug in conven‐ tional and high dosages. However, more recent work highlighted as an immunostimulatory and/or antiangiogenic agent at low dosages, openning up novel indication in the field of cancer immunotherapy. In recent reports, cyclophosphamide administration in tumor-bearing mice induced pre-apoptotic surface translocation of calreticulin on tumor cells [101], which serves as an "eat-me" signal for phagocytes [102] and the release of high-mobility group box1 (HMGB1) protein in the extracellular milieu [101], which constitutes a "danger signal" triggering activation of the DC processing machinery [103]. These events are prerequisites for adequate engulfment of tumor apoptotic material and optimal CD8+ T cell cross-priming by DCs [102, 103].

HSPs are intracellular chaperones for many proteins, but they can also be expressed on the cell surface or even be released under stress conditions [104, 105]. HSP acts as an adjuvant in initiating the activation of DCs or as protein vehicle to facilitate the presentation of antigen peptides to T cells. Spisek et al. [106] reported that uptake of myeloma cells by DCs after tumor cell death induced by bortezomib leads to the induction of antitumor immunity and enhances DC-mediated tumor immune response, indicating the probability mechanism due to the expression of HSP90 on the surface of dying cells, thereby facilitating the activation of DCs in response to dying tumor cells. Our study also found that HSPs released from dying tumor cells, which were induced by a combination of the selective JAK/STAT3 inhibitor JSI-124 and proteasome inhibitor bortezomib, act on tumor cells to recover DC dysfunction and to induce cytokine and chemokine production from DCs, resulting in generation of potent myelomaspecific CTL response against myeloma cells.

#### **5.4. Possible combination DCs and other approaches**

In the presence of regulatory and suppressive environment, it is very difficult to elicit or induce effective immune response after DC vaccination in cancer patients. To improve the clinical outcomes, DC vaccines need to be combined, in particular for patients at advanced stages, with other approaches that offset the suppressive tumor environment [107]. It has been known that the specific depletion of CD4+ CD25+ Treg cells by anti-CD25 antibodies increases the efficiency of the anti-tumor immune response of tumor-bearing animals, although the tumors are not completely rejected [108]. An increased number of CD4+ CD25+ FoxP3+ regulatory T cells have been demonstrated in patients with MM [22, 109]. Depletion of Treg may have resulted in improved response to tumor vaccine in animal models and a clinical study. In addition, blocking antibodies or soluble receptors were exploited for the blockade of suppressive cytokines in the tumor microenvironment, such as IL-10 [110], IL-13 [111], TGF-β [112] and VEGF [113]. Such strategies can be used to block immune-inhibitory signals in lymphocytes as illustrated by anti-CTLA-4 [114] and/or anti-PD1 [115] or to block their ligands expressed on tumors.

the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy; grant no. A000200058 from the Regional Industrial Technology Development program of the Ministry of Knowledge and Economy; grant no. 1120390 from the National R&D Program for Cancer Control, Ministry for Health and Welfare; grant no. 2011-0030034 from Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology

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189

, Hyun Ju Lee1,2, Sung-Hoon Jung1,2 and

1 Research Center for Cancer Immunotherapy, Chonnam National University Hwasun

2 Department of Hematology-Oncology, Chonnam National University Hwasun Hospital,

[1] Kyle RA, Rajkumar SV. Multiple myeloma. N Engl J Med 2004; 351: 1860-1873.

[3] Attal M, Harousseau JL. The role of high-dose therapy with autologous stem cell

[4] Lonial S, Cavenagh J. Emerging combination treatment strategies containing novel agents in newly diagnosed multiple myeloma. Br J Haematol 2009; 145: 681-708.

[5] Perez-Simon JA, Martino R, Alegre A et al. Chronic but not acute graft-versus-host disease improves outcome in multiple myeloma patients after non-myeloablative al‐

[6] Harrison SJ, Cook G, Nibbs RJ, Prince HM. Immunotherapy of multiple myeloma: the start of a long and tortuous journey. Expert Rev Anticancer Ther 2006; 6:

[7] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature

3 Vaxcell-Bio Therapeutics, Hwasun, Jeollanamdo, Republic of Korea

[2] Sirohi B, Powles R. Multiple myeloma. Lancet 2004; 363: 875-887.

logeneic transplantation. Br J Haematol 2003; 121: 104-108.

support in the era of novel agents. Semin Hematol 2009; 46: 127-132.

(MEST), Republic of Korea.

Je-Jung Lee1,2,3, Youn-Kyung Lee3

Hospital, Jeollanamdo, Republic of Korea

Thanh-Nhan Nguyen-Pham1,2

Jeollanamdo, Republic of Korea

**Author details**

**References**

1769-1785.

1998; 392: 245-252.

Another strategy to improve DC vaccination is combination approach with other immune cells, including adoptive T cells or NK cells. In adoptive T-cell transfer, one can seek to modulate the number of regulatory T cells, and transfer a population of activated effector cells. The combination of DC vaccination and adoptive T-cell transfer led to a more robust antitumor response than the use of each treatment modality [116]. These findings illuminate a new potential application for DC vaccination in the *in vivo* stimulation of adoptively transferred T cells. Therefore, combining active and passive immunotherapies in the treatment of MM may enhance the efficacy of tumor vaccine in the future.

#### **6. Future perspectives**

Progress in understanding DC biology in MM patients and the recruitment of suppressive cells of the adaptive and innate immune system in antitumor immunity of cellular immunotherapy is leading to new concept which aims at improved immune and clinical outcomes in MM. The new generation of DCs may be a potential vaccine therapy for inducing the rate of tumor responses and prolonging survival of patients with MM. Furthermore, information from studies that combine DC vaccine with other therapies, including chemotherapy, radiation therapy, molecular target agents, other immunotherapy (adaptive T cells or NK cells), or adjuvants will have high impact on enhancing therapeutic immunity in MM by simultaneously enhancing the potency of immune responses and offsetting immunoregulatory pathways.

#### **Acknowledgements**

This study was financially supported by grant no. 2011-0005285 from General Researcher Program Type II of the National Research Foundation of Korea; grant no. RTI05-01-01 from the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy; grant no. A000200058 from the Regional Industrial Technology Development program of the Ministry of Knowledge and Economy; grant no. 1120390 from the National R&D Program for Cancer Control, Ministry for Health and Welfare; grant no. 2011-0030034 from Leading Foreign Research Institute Recruitment Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST), Republic of Korea.
