An update on new adoptive immunotherapy strategies for solid tumors with cytokine-induced killer cells.

Review

1.

Introduction

2.

Improving CIK cell proliferation and cytotoxicity through combination with additional cytokines

Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Laval on 06/11/14 For personal use only.

3.

Adoptive CIK cell therapy in combination with oncolytic viruses

4.

Combining CIK cell and dendritic cell immunotherapy: in vitro, in vivo and clinical studies

5.

Targeted CIK cell therapy

6.

CIK cell therapy in combination with antiangiogenic drugs

7.

Clinical studies on CIK cell therapy combined with minimally invasive therapies

8.

Clinical studies on CIK cell therapy combined with surgery, chemo- and cytokine therapy

9. 10.

An update on new adoptive immunotherapy strategies for solid tumors with cytokine-induced killer cells Clara E Ja¨kel & Ingo GH Schmidt-Wolf† †

University Hospital Bonn, Center for Integrated Oncology (CIO), Bonn, Germany

Introduction: Cytokine-induced killer (CIK) cells are mainly CD3+CD56+ NKT cells exhibiting non-MHC-restricted cytotoxicity against a broad range of tumors. Much research is going on to improve CIK cell effectivity and to evaluate the clinical benefit of different combinations with conventional therapies. Areas covered: This review provides an update on in vitro/in vivo studies and clinical trials applying CIK cells for the treatment of solid tumors. This comprises attempts using additional cytokines, genetic engineering and combinations with different conventional and modern therapies. Expert opinion: Since our last review, much effort has been made to improve CIK cell cytotoxicity and clinical effectivity. Targeted CIK cell therapy and combinations of CIK cells with antiangiogenic drugs or oncolytic viruses are examples of recent outstanding achievements in the field of adoptive CIK cell therapy. The clinical application of CIK cells in combination with conventional therapies, especially, obtained promising results. However, the best combination and the optimal therapy schedule have yet to be defined. Keywords: adoptive immunotherapy, cytokine-induced killer cells, dendritic cells, NKT cells, solid tumors

Conclusion Expert opinion

Expert Opin. Biol. Ther. (2014) 14(7):905-916

1.

Introduction

Although new tumor treatment strategies are constantly developed, cancer recurrence and mortality rates remain high. Adoptive immunotherapy, as adjuvant or alternative treatment, is anticipated to be an innovative approach to treat any type of malignancy. The general concept of immunotherapy is to stimulate the patient’s immune system ex vivo or in vivo in order to induce an antitumor immune response and to restore the patient’s immune status. Cytokine-induced killer (CIK) cells meet all requirements for application in adoptive immunotherapy. These cells are generated from peripheral blood lymphocytes through the timed application of IFN-g, monoclonal antibody against CD3 (anti-CD3) and IL-2. The protocol for induction of CIK cells can vary between different research groups; sometimes IL-1 is added as well. The addition of IFN-g 24 h before the addition of anti-CD3 and IL-2 is critical for the induction of the maximal cytotoxicity. At the beginning of cultivation, IFN-g provides activating signals to remnant monocytes. These activated monocytes provide contact-dependent (CD58/LFA-3) and soluble (IL-12) factors important for the expansion of CD56+ cells [1,2]. The combination of anti-CD3 and IL-2 is essential for the proliferation of CIK cells [3]. Within the expanded CIK cell culture, which is a heterogeneous population of T lymphocytes, CD3+CD56+ double-positive cells are the most cytotoxic cells [4-6]. 10.1517/14712598.2014.900537 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted

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C. E. Ja¨kel & I. G. H. Schmidt-Wolf

Article highlights. . .

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Cytokine-induced killer (CIK) cell cytotoxicity can be improved through cultivation with additional cytokines. The combination of CIK cells and oncolytic viruses can achieve promising results through several different approaches. Combined dendritic cell-CIK cell immunotherapy has great potential in cancer therapy. Targeting CIK cells with bispecific antibodies or chimeric antigen receptors to tumor cells can increase CIK cell efficacy. Combinations of CIK cells with minimally invasive therapies, surgery and chemotherapy could improve the clinical outcome of cancer patients. CIK cell therapy is a promising therapeutic approach for patients with solid malignancies; still much research is needed to fully explore the potential of CIK cell therapy.

This box summarizes key points contained in the article.

They derive from proliferating CD3+CD56- cells and, in contrast to the bulk CIK cell population, show minimal alloreactivity across HLA barriers [5-7]. Interestingly, they originate from the subsets of CD4-CD8+ and CD4-CD8cells, but not from CD4+CD8- cells [1]. The cytotoxicity of these CD3+CD56+ cells is non-MHC-restricted, perforinmediated and induced via the natural killer group 2 member D (NKG2D) cell-surface receptor [8,9]. NKG2D ligands, for example, MHC class I-related chain (MIC) A/B and UL-16 binding protein 1 -- 4, are overexpressed on both solid and hematologic tumor cells, turning these cells into a favored target of CIK cells [10,11]. It was shown that, during CIK cell therapy, a high expression of MICA in patients with advanced NSCLC correlates with a longer median overall survival (OS). This is particularly interesting as a high expression of MICA usually indicates a poor prognosis, but might in this way be a prospective inclusion criterion for CIK cell therapy [12]. Interestingly, the clinical effectivity of CIK cell therapy in cancer patients seems not to be merely dependent on CD3+CD56+ cells. Pan et al. provide a retrospective clinical study evaluating the association between the CIK cell phenotype and the therapeutic efficacy in 121 hepatocellular carcinoma (HCC), 74 lung and 42 colorectal cancer patients [13]. In the end, different phenotypic subsets of the CIK cell population could be associated with improved clinical outcomes in different types of cancer -- a high ratio of CD3+CD56+ cells was associated with a higher OS in lung and colorectal cancer and a high ratio of CD3+CD8+ cells was associated with improved OS in all patients. Similarly, a high ratio of CD3-CD56+ NK cells was associated with a lower OS in lung and colorectal cancer and a high ratio of CD3+CD4+ cells was associated with poorer OS only in colorectal cancer. Therefore, high numbers of CD3+CD56+ and CD3+CD8+ cells seem to be critical for the induction of an effective antitumor response in cancer patients. 906

Apart from their advantageous cytotoxic profile, it has recently been discovered that CIK cells are even able to kill autologous putative melanoma and sarcoma cancer stem cells (CSCs) [14,15]. CSCs are tumor cells with stemness characteristics causing relapse and drug resistance [16]. Therefore, targeting CSCs seems to be indispensable to efficiently eradicate a tumor and prevent recurrence. In the last decade, much effort has been made to optimize cytotoxicity and application of CIK cells. In the following sections, we will provide an update on our last review about new adoptive immunotherapy strategies for solid tumors with CIK cells [17].

Improving CIK cell proliferation and cytotoxicity through combination with additional cytokines

2.

The CIK cell culture is a very heterogeneous cell population, which comprises not only cytotoxic cells but, among others, also regulatory T cells (Treg). Because Treg cells are known to inhibit antitumor immunity, many efforts have been made to minimize the number of Treg cells within the CIK cell culture. IL-6 inhibits the generation of FoxP3+ Treg cells and is therefore an appealing cytokine to enhance CIK cell cytotoxicity [18]. In fact, the addition of IL-6 every 2 -- 3 days during CIK cell generation decreased the proportion of Treg cells within the CIK cell population [19]. But IL-6 showed even more beneficial effects on CIK cells: within the IL-6-treated CIK cell culture, the proliferation rate (p < 0.001) and the number of CD3+CD56+ cells (p = 0.036) increased significantly compared with regular CIK cells. As a result, the cytotoxicity of IL-6-treated CIK cells toward HCC cells was also significantly higher. Nevertheless, the cytotoxicity of these CIK cells was as minimal toward freshly isolated fibroblasts as that of regular CIK cells and peripheral blood mononuclear cells (PBMC). IL-12, naturally expressed by antigen-presenting cells, has antiangiogenic properties and gives activating signals to cytotoxic T cells, thereby inducing immune responses. CIK cells have already been generated using IL-12 instead of IL-2 [20]. The cytotoxicity of both CIK cell cultures was similar. However, cells stimulated with IL-12 had a lower proliferation rate combined with a higher necrosis rate. Due to its high toxicity in clinical application, IL-12 was tested in lower doses in combination with other therapeutic approaches, for example, CIK cells [21]. Compared with either therapy alone, the concomitant therapy with IL-12 and CIK cells in a murine breast cancer model led to an increased antitumor effect -- with full tumor remission and long-term protection in 75% of the mice. Moreover, short-term cultured CIK cells, which showed no cytotoxicity when applied alone, exhibited full antitumor activity in vivo when combined with IL-12. Several studies proved that the stimulation of CIK cells with IL-15 instead of or along with IL-2 can improve CIK

Expert Opin. Biol. Ther. (2014) 14(7)


An update on new adoptive immunotherapy strategies for solid tumors with cytokine-induced killer cells

cell cytotoxicity against hematologic and solid malignancies [22-25]. During stimulation with IL-15, the expression of Toll-like receptor 4 (TLR4) is upregulated and knock-down of the TLR4 results in considerably compromised cytotoxicity [26]. IL-15 increases the proliferative rate and the expansion of CD3+CD56+ cells within the CIK cell population, while it decreases the number of Treg cells [24,25]. Accordingly, IL-15-stimulated CIK cells exhibit enhanced cytotoxic activity in vitro and in vivo [22-25]. Finally, IL-21 was currently evaluated for its potential to enhance CIK cell effectivity [27,28]. The proliferative rate of CIK cells was not increased through IL-21 application, but the number of CD3+CD56+ cells. Along with an improved cytotoxicity, an increase of intracellular perforin and granzyme B was shown. Furthermore, the release of IFN-g and TNF-a increased, while the expression of granzyme A, TNF-b and NKG2D remained on a similar level.

Adoptive CIK cell therapy in combination with oncolytic viruses

3.

Oncolytic viruses can be engineered with the E1A gene, which is essential for viral replication, being regulated by the E2F-1 promoter. The gene encoding the transcription factor E2F-1 is overexpressed in many solid tumors, resulting in selective viral replication in these cancer cells. Yan et al. used an E2F-1 promoter-regulated oncolytic adenovirus (Ad-E2F) in combination with CIK cell adoptive therapy [29]. This study proved an increased E2F-1 expression in rectal cancer tissue -- a prerequisite for the effective application of the Ad-E2F. The designed oncolytic virus selectively killed colorectal cancer cells, while it had no significant effect on CIK cells. The combinational therapy of Ad-E2F and CIK cells was capable of producing a stronger antitumor effect than the application of either therapy alone. As mentioned above, Helms et al. reported an increased therapeutic effect of CIK cells when applied along with IL-12 [21]. Yang et al. extended this idea combining adoptive CIK cell therapy with an oncolytic adenovirus expressing IL-12 [30]. The oncolytic adenoviral system was engineered to replicate only in tumor cells and, during viral replication, the introduced IL-12 gene should be expressed. The same adenoviral system harboring a gene encoding the green fluorescent protein (GFP) instead of IL-12 and a replicationdefective IL-12-adenovirus were used as controls. Comparing the effects of the IL-12-bearing adenovirus (AdCN205-IL12) and the control viruses, the AdCN205-IL12 revealed a higher tumor cell-specific expression of IL-12. However, the cytotoxicity was the same as that of the GFP control virus, indicating that IL-12 lacks a direct effect on tumor cell survival. This study also reported an increase in CIK cell tumor infiltration and antitumor activity through combination with AdCN205-IL12. In vivo, the combinational therapy led to tumor regression and long-term survival in an established murine liver tumor model. Interestingly, also the application of CIK

cells along with the GFP-expressing adenovirus was superior to either therapy alone. This indicates an increased therapeutic effect through combination of the oncolytic virus and CIK cell therapy, with an even more enhanced effect when combined with IL-12. Another approach combining virotherapy and CIK cell therapy included an oncolytic measles virus (MV) [31]. MVs have already been successfully delivered to cancer cells using T cells, but CIK cells might be superior due to their intrinsic antitumor activity [32]. Liu et al. showed that CIK cells are susceptible to MV infection and are capable of transferring an MV infection to myeloma cells [31]. The expression of NKG2D was not influenced by viral infection and the cytotoxicity of MV-CIK cells was higher than that of an MV-infected myeloma cell line. Compared with uninfected CIK cells, MV-infected CIK cells prolonged survival of mice with disseminated myeloma. In order to enhance the antitumor effect, the MV-infected CIK cell therapy was furthermore combined with ionizing radiation (XRT). Increasing doses of radiation induced increasing levels of MICA and MICB, probably contributing to the enhanced therapeutic effect when combining MV-infected CIK cells with XRT.

Combining CIK cell and dendritic cell immunotherapy: in vitro, in vivo and clinical studies

4.

Cellular interactions between CIK cells and dendritic cells (DCs) lead to an increase of cell surface markers important for cytotoxicity, finally resulting in a higher cytolytic CIK cell activity [33]. DC--CIK immunotherapy has been shown to be effective without causing major side effects, and therefore, CIK cell adoptive therapy is frequently combined with DC vaccination [34,35]. Wang et al. provide an in vitro study combining CIK cells and DCs for the treatment of retinoblastoma (RB) [36]. DCs were pulsed with complete tumor antigens and co-cultured with CIK cells. By this means, CIK cell proliferation and cytotoxicity toward two RB cell lines, one of them being resistant to carboplatin chemotherapy, could be increased. Importantly, these CIK cells showed little toxicity against a normal retina cell line. Another approach of DC--CIK immunotherapy includes the tyrosine kinase inhibitor sunitinib [37]. DCs were incubated with sunitinib for 48 h prior to co-culture with CIK cells. CIK cell cytotoxic activity was significantly increased after co-culture with sunitinib-treated DCs compared with CIK cells co-cultured with untreated DCs or CIK cells directly treated with sunitinib. Examination of subpopulations within the differently treated CIK cell cultures revealed no significant changes in CD3+CD56+, Th17, or Treg proportions. However, the authors state a polarization of CD3+CD56+ cells toward Th1 differentiation through the increased expression of IFN-g and T-bet.


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Several studies examine the value of the combination of DC--CIK immunotherapy with chemotherapy. A single intraperitoneal subtoxic cisplatin dose (5 mg/kg) was found to strengthen tumor homing and antitumor activity of CIK cells in a murine melanoma model [38]. CIK cells co-cultivated with antigen-pulsed DCs were five times more cytotoxic on RB cells pretreated with a low dose of carboplatin than on untreated cells [39]. During a clinical study, 122 patients with advanced NSCLC were treated with chemotherapy (Navelbine + Cisplatin) and 61 of these patients received additional DC-activated CIK cell transfusions 1 month after completion of chemotherapy [40]. DC-activated CIK cell therapy in combination with chemotherapy significantly (p < 0.05) increased 1- and 2-year OS rates (study group: 57.2 and 27.0%; control group: 37.3 and 10.1%). Similarly, a group of 60 advanced NSCLC patients was treated with platinumbased chemotherapy and, after reaching stable disease, divided into a control and a study group [41]. Patients in the study group were given additional DC--CIK therapy. Application is described as safe and effective, significantly (p < 0.05) prolonging progression-free survival (PFS). In a different clinical study, NSCLC patients treated with navelbine-platinum chemotherapy were additionally treated with autologous DC--CIK transfusions, either twice every 30 days or more than twice every 30 days [42]. The 1-, 2and 3-year OS rates in the patient group receiving more transfusions were significantly (p = 0.037) higher. The time-to-progression was also significantly (p = 0.034) prolonged. Therapy with autologous tumor lysate-pulsed DCs and CIK cells applied after surgical tumor resection was also able to significantly (p < 0.01) prolong OS rates and decrease recurrence rates in patients with locally advanced renal cell carcinoma (RCC) [43]. Qiu et al. conducted two clinical studies with a-galactosyl (a-Gal) epitope-activated DCs and CIK cells [44,45]. a-Gal epitopes were synthesized on HCC and pancreatic tumor cells, respectively, and incubated with natural human anti-Gal IgG, resulting in opsonization and phagocytosis by DCs. These DCs were co-cultured with CIK cells and this cell mixture was used for infusions. HCC patients treated with these DC--CIK injections along with conventional therapies survived significantly (p = 0.00121) longer than control patients only treated with conventional therapies [44]. Unfortunately, this control group is missing in the second study [45]. Following immunotherapy, expression of IFN-g by PBMC and increased levels of CD8+, CD45RO and CD56+ cells could be detected in both studies. The above-mentioned clinical studies using DC--CIK immunotherapy are summarized in Table 1. 5.

Targeted CIK cell therapy

CIK cells have been armed with several bispecific antibodies (BsAb) in order to increase their therapeutic effect. Du et al. 908

used a carcinoembryonic antigen (CEA)/CD3-BsAb to treat an established, subcutaneous gastric cancer in a murine model via different infusion routes [46]. After intravenous and intraperitoneal infusion, only a few CIK cells could be detected at the tumor site and tumor growth was reduced only directly after cell application. Peritumoral injection resulted in a high number of CIK cells within the tumor tissue and a higher inhibition of tumor growth. Furthermore, the antitumor activity of CIK cells and cytotoxic T lymphocytes (CTL), which were generated by co-incubation with antigen-pulsed DCs, was compared in vitro and in vivo. After peritumoral injection, significantly more BsAb-CIK cells than standard CIK cells accumulated within the tumor. Still, DC-stimulated CTL revealed the strongest in vitro and in vivo cytotoxicity. CIK cells have also been targeted to a gastric cancer cell line using an EGFR/CD3 BsAb [47]. In vitro cytotoxicity and tumor growth reduction in a murine model was highest using CIK cells armed with the BsAb compared with the controls. Recently, CIK cells have been targeted with an antiCD133/CD3 BsAb to CSCs highly expressing CD133 [48]. Compared with the controls, cytotoxicity of BsAb-CIK cells on cancer cells expressing high CD133 was significantly (p < 0.05) stronger. This significance was limited to cells expressing high CD133 as the cytotoxic effect on tumor cells expressing low levels of CD133 was similar among BsAb-CIK, CD3-CIK and regular CIK cells. A different approach to target CIK cells to tumor cells involves the genetic engineering of CIK cells with a chimeric antigen receptor (CAR) with an antibody-defined specificity against a target antigen. Schlimper et al. provide data on CIK cells engineered with a CAR directed against CEA [49]. These CIK cells secreted higher levels of pro-inflammatory cytokines in the presence of CEA-positive colon carcinoma cells than in the presence of CEA-negative cells. Compared with regular CIK cells, activation of CAR-engineered CIK cells against autologous primary tumor cells from colon carcinoma patients was increased. Remarkably, ‘super-stimulation’ by a CD28-z-OX40 CAR can lead to activation-induced cell death in CIK cells [50].

CIK cell therapy in combination with antiangiogenic drugs

6.

Hypothesizing that the tumor vasculature and the hypoxic tumor microenvironment might impair the effectiveness of CIK cell therapy, Shi et al. provide a study applying recombinant human endostatin along with CIK cells [51]. Hypoxia was found to significantly inhibit the proliferation, cytotoxicity and migration of CIK cells in vitro. Moreover, hypoxia inhibited the infiltration of CIK cells into tumor parenchyma ex vivo. Endostatin was able to normalize the tumor vasculature and decrease the hypoxic area. The combinational treatment with endostatin increased CIK cell tumor infiltration and hindered the accumulation of suppressive immune cells



Chemotherapy; patients in the study group received additional DC--CIK immunotherapy Chemotherapy; patients in group I (n = 30) received additional DC--CIK therapy twice every 30 days; patients in group II (n = 30) received additional DC--CIK therapy more than twice every 30 days Surgery; patients in the study group (n = 46) received adjuvant DC--CIK immunotherapy; patients in the IFN group (n = 46) received adjuvant IFN-g therapy; patients in the control group (n = 45) received no adjuvant treatment

Surgery and radio-, chemo-, or interventional therapy; patients in the study group (n = 9) received additional a-Gal epitope-pulsed DC--CIK therapy All patients were inoperable and received a-Gal epitope-pulsed DC--CIK therapy

60 patients, NSCLC

60 patients, NSCLC

137 patients, RCC

18 patients, HCC

14 patients, pancreatic carcinoma

[41]

[42]

[43]

[44]

[45]

Side effects were less in the study group than in the IFN group; recurrence/metastasis rate was significantly lower in study and IFN group than in control group (0/6.7/26.7%, respectively, p < 0.01); 5-year OS rates were significantly higher in study and IFN group than in control group (100/100/88.9%; p < 0.01); there were no significant differences between study and IFN group (p > 0.05) No serious side effects after DC--CIK therapy; OS: 17.1/ 10.1 (p = 0.00121); IFN-g secretion by PBMCs increased, while serum AFP decreased after DC--CIK therapy No serious side effects; increased IFN-g secretion by PBMCs

1-, 2- and 3-year OS rates (group I/group II): 56.7/63.3%; 13.3/30.0%; 6.7/23.3% (p < 0.05)

DC--CIK therapy prolonged PFS: 3.20/2.56 months (p < 0.05)

1- and 2-year OS: 57.2/37.3 and 27.0/10.1% (p < 0.05)

Results (immunotherapy/control group)

DC--CIK therapy with a-Gal epitope-pulsed DC is safe and feasible

DC--CIK therapy with a-Gal epitope-pulsed DC is safe and effective

Adjuvant DC--CIK immunotherapy is safe and can prolong OS and prevent recurrence and metastasis

Chemotherapy in combination with DC--CIK immunotherapy leads to improved clinical outcomes in patients with NSCLC DC--CIK therapy is safe and has a potential benefit for NSCLC patients Chemotherapy along with DC--CIK therapy was safe and effective; higher numbers of immunotherapy cycles increased the beneficial effect of DC--CIK immunotherapy

Conclusions

AFP: a-fetoprotein; CIK: Cytokine-induced killer; DC: Dendritic cell; HCC: Hepatocellular carcinoma; OS: Overall survival; PBMCs: Peripheral blood mononuclear cells; PFS: Progression-free survival; RCC: Renal cell carcinoma; a-Gal: a-galactosyl.

Chemotherapy; patients in the study group (n = 61) received additional DC--CIK immunotherapy

Therapy

61 paired patients, NSCLC

Number of patients, tumor type

[40]

Study

Table 1. Clinical studies on DC--CIK immunotherapy.



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within the tumor tissue. In conclusion, CIK cell therapy could be improved when combined with endostatin as antiangiogenic therapy. Similarly, the antiangiogenic antibody bevacizumab has been used to improve CIK cell adoptive therapy [52]. Mice bearing a NSCLC were treated with bevacizumab and CIK cells. Bevacizumab reduced the hypoxic area within the tumor tissue and normalized the tumor vasculature. As in the study before, the combinational therapy improved CIK cell tumor infiltration and antitumor efficacy.

7. Clinical studies on CIK cell therapy combined with minimally invasive therapies

Transarterial chemoembolization (TACE) combined with radiofrequency ablation (RFA) has successfully been applied for the treatment of HCC. In order to further improve the patients’ long-term prognosis, CIK cell adoptive therapy may be applied additionally as discussed in our previous review [17]. Up to now, further clinical trials have been conducted to evaluate the benefit of CIK cell therapy after TACE/RFA. Wang et al. provide data of 76 HCC patients treated with TACE/RFA; 38 of these patients received additional autologous CIK cell transfusions [53]. There were no severe side effects in both treatment groups, and the median follow-up time ranged between 10 and 88 months. The 1-, 3- and 5-year disease-free survival (DFS) was significantly longer in the CIK-treated group than in the control group (p = 0.001; 28 vs 22 months). Still, the OS was not significantly different between the two groups. At the same cancer center, a retrospective study was analyzed including 89 patients treated with TACE/RFA and 85 patients treated with additional CIK cell adoptive therapy [54]. After a median follow-up of 78 months, the PFS was significantly (p < 0.001) longer in the CIK-treated group (17.0 months) than in the control group (10.0 months). The 1-year OS rates were not significantly (p = 0.241) different between the groups, but the 3-, 5- and 10-year OS rates were significantly (p £ 0.005) higher for patients treated with CIK cells. Cryoablation is a local therapy applied for the treatment of several solid tumors. In two clinical studies, the combination of cryotherapy with DC--CIK cell therapy attained encouraging results [55,56]. In patients with metastatic HCC, cryo-immunotherapy led to a significantly (p < 0.05) better median OS compared with either therapy alone and multiple treatments were superior to a single treatment (p < 0.05) [55]. Similarly, the combination of cryotherapy, DC--CIK immunotherapy and chemotherapy (platinum-based) was applied to treat metastatic NSCLC [56]. This combination achieved a significantly (p < 0.001) longer OS than cryo-chemotherapy, cryo-immunotherapy, chemo-immunotherapy and chemotherapy alone. 910

Clinical studies on CIK cell therapy combined with surgery, chemo- and cytokine therapy

8.

Wang et al. evaluated the effect of CIK cell therapy combined with IL-2 application on clinical outcome and level of peripheral myeloid-derived suppressor cells (MDSCs) in 29 patients with metastatic RCC [57]. MDSCs promote angiogenesis and tumor cell survival and might therefore restrict clinical efficacy of immunotherapy. Interestingly, survival of patients with lower levels of MDSCs at study entry was significantly (p = 0.013) prolonged. In the beginning, the 29 patients displayed increased levels of MDSCs compared with agematched healthy controls. Following CIK therapy, a significant (p < 0.01) decrease of MDSCs could be detected in the patients’ peripheral blood. Adoptive CIK cell transfer is often applied as adjuvant therapy following surgical resection of the tumor. Accordingly, 151 postoperative gastric cancer patients were divided into a control group receiving no additional treatment and an immunotherapy group treated with adjuvant CIK cell therapy [58]. Immunotherapy significantly (p = 0.044) prolonged 5-year DFS (28.3 vs 10.4%) but not 5-year OS (32.4% in immunotherapy group vs 23.4% in control group, p = 0.071). Importantly, the 5-year OS was significantly (p = 0.045) improved after immunotherapy in patients with intestinal-type tumors (46.8% in immunotherapy group vs 31.4% in control group). In a different study, 165 patients with advanced gastric carcinoma, who had undergone surgical resection and chemotherapy, were retrospectively analyzed [59]. Fifty-three of all patients received chemotherapy combined with CIK cell therapy. The 5-year OS rate was significantly (p = 0.014) higher in the CIK-treated group than in the control group (56.6 vs 26.8%). Moreover, the 5-year PFS rate was also significantly (p = 0.026) prolonged for patients who were treated with adjuvant CIK cell therapy (49.1 vs 24.1%). Liu et al. combined CIK cell therapy with FOLFOX4 chemotherapy for the postoperative treatment of gastric carcinoma [60]. Compared with the control group, the 1-, 2- and 3-year recurrence rates were significantly (p < 0.05) reduced in the immunotherapy group while the survival rates were significantly (p < 0.05) improved. In a retrospective study including 410 HCC patients, adjuvant CIK cell therapy could significantly (p = 0.0007) improve OS rates compared with surgery alone [61]. Survival was also related to the number of CIK cell therapy cycles as patients who received more than eight cycles survived significantly (p = 0.0272) longer than patients who received less. Similarly, a prospective study with 66 HCC patients receiving standard therapy (surgery, TACE, or best support care) and 66 patients receiving additional CIK cell therapy was conducted [62]. The 1-, 2- and 3- year OS was significantly (p £ 0.005) higher for patients treated with additional CIK cell therapy.


Gastrectomy; patients in the study group (n = 74) received adjuvant CIK cell therapy Gastrectomy and chemotherapy; patients in the study group (n = 53) received adjuvant CIK cell therapy Gastrectomy and FOLFOX4 chemotherapy; patients in the study group (n = 51) received additional CIK cell therapy Surgery; patients in the study group (n = 204) received at least four cycles of adjuvant CIK cell therapy

Surgery, TACE or BSC; patients in the study group (n = 66) received additional CIK cell therapy Chemotherapy; patients in the study group (n = 87) received additional CIK cell therapy

151 patients, advanced gastric cancer 165 patients, advanced gastric cancer

98 patients, gastric cancer

410 patients, HCC

132 patients, HCC

87 paired patients, NSCLC

[58]

[60]

[61]

[62]

[63]


Among early-stage patients: 3-year PFS and median PFS were not significantly different between study and control group, but 3-year OS and median OS were higher in study group (p < 0.05); among advanced-stage patients: 3-year PFS and OS higher in study group (p < 0.001), median PFS and OS longer in study group (p £ 0.001)

1-, 2- and 3-year OS: 74.2/50.0%; 53.0/30.3%; 42.4/24.2% (p £ 0.005)

5-year OS: 56.6/26.8% (p = 0.014); 5-year PFS: 49.1/24.1% (p = 0.026); median OS: 96/32 months (p = 0.028); median PFS: 36/23 months (p = 0.003) 1-, 2- and 3-year recurrence rates: 5.9/ 25.5%; 17.6/36.2%; 23.5/48.9% (p < 0.05); 1-, 2- and 3-year survival rates: 98.0/93.6%; 92.2/78.7%; 72.5/ 59.6% (p < 0.05) OS rates higher in study group than in control group (p < 0.001); survival of patients who received ‡ 8 cycles of CIK cells longer (p < 0.05)

4 PR (13.8%), 18 SD (62.1%), 7 PD (24.1%); 1-year survival 82.8% (24 patients); peripheral blood MDSC levels, which were elevated in most patients at study entry, decreased after CIK cell therapy 5-year OS: 32.4/23.4% (p = 0.071); 5-year DFS: 28.3/10.4% (p = 0.044)

Results (immunotherapy/control group)

CIK cell therapy could improve efficacy of chemotherapy and prolong OS in patients with NSCLC

Adjuvant CIK cell therapy may be effective to improve the outcomes of patients with HCC; ‡ 8 cycles of CIK cell infusions may be essential to achieve best results CIK cell therapy can prolong OS in patients with HCC

FOLFOX4 combined with CIK cell therapy significantly prolonged survival and reduced recurrence in gastric cancer patients

Adjuvant CIK cell therapy prolongs DFS of patients with advanced gastric cancer Adjuvant CIK cell therapy may prolong survival of patients with stage II--III gastric carcinoma

CIK cell therapy can induce mRCC regression; MDSCs can serve as potential prognostic marker in CIK cell therapy

Conclusions

BSC: Best supportive care; CIK: Cytokine-induced killer; DFS: Disease-free survival; HCC: Hepatocellular carcinoma; IFN-a2a : Interferon-a2a; MDSC: Myeloid-derived suppressor cells; mRCC: Metastatic renal cell carcinoma; NSCLC: Non-small cell lung cancer; OS: Overall survival; PD: Progressive disease; PFS: Progression-free survival; PR: Partial response; SD: Stable disease; TACE: Transarterial chemoembolization.

[59]

CIK cell therapy + IL-2

Therapy

29 patients, mRCC

Number of patients, tumor type

[57]

Study

Table 2. Clinical studies on CIK cells combined with surgery, chemo- and cytokine therapy.



911

912

CIK cell therapy could improve the prognosis of patients with mRCC, especially with increasing CIK cell cycle count 3-year PFS: 18/12% (p = 0.031); median PFS: 12/8 months (p = 0.024); 3-year OS 61/23%(p < 0.001); median OS: 46/19 months (p < 0.001); increasing CIK cell cycle count was associated with longer PFS and OS (p < 0.001) [65]

BSC: Best supportive care; CIK: Cytokine-induced killer; DFS: Disease-free survival; HCC: Hepatocellular carcinoma; IFN-a2a : Interferon-a2a; MDSC: Myeloid-derived suppressor cells; mRCC: Metastatic renal cell carcinoma; NSCLC: Non-small cell lung cancer; OS: Overall survival; PD: Progressive disease; PFS: Progression-free survival; PR: Partial response; SD: Stable disease; TACE: Transarterial chemoembolization.

Adjuvant CIK cell therapy can prolong DFS in patients with colorectal cancer 2-year DFS: 59.65/29.35% (p < 0.05)

Surgery, chemotherapy; patients in the study group (n = 21) received at least one cycle of additional CIK cell therapy Study group (n = 74): CIK cell therapy; control group (n = 74): IL-2/IFN-a2a therapy 96 patients, colorectal cancer 148 patients, mRCC [64]

Number of patients, tumor type Study

Table 2. Clinical studies on CIK cells combined with surgery, chemo- and cytokine therapy (continued).


Conclusions Results (immunotherapy/control group) Therapy


Li et al. conducted a Phase II clinical study to assess the efficiency of postoperative CIK cell therapy combined with chemotherapy for patients with early-stage and advancedstage NSCLC [63]. Between the immunotherapy and the control group, there was no statistical significance in 3-year PFS and median PFS among early-stage patients. Nevertheless, the 3-year OS and median OS of patients with early-stage tumors was significantly (p = 0.049 and p = 0.006, respectively) improved by CIK cell therapy. Within the immunotherapy group, OS and PFS were significantly (p £ 0.001) prolonged for patients with advanced-stage disease. Another retrospective study included 96 colorectal cancer patients who underwent surgery and adjuvant chemotherapy; 21 of these patients received additional CIK cell transfusions [64]. CIK cell therapy was able to prolong DFS in colorectal cancer patients as it was significantly (p = 0.034) longer for patients in the immunotherapy group than in the control group. CIK cell therapy has also been compared with cytokine treatment with IL-2 and IFN-a2a [65]. The prospectively randomized clinical study included 148 patients with metastatic RCC who either underwent CIK cell immunotherapy (74 patients) or cytokine treatment (74 patients). The 3-year PFS was significantly (p = 0.031) longer in the immunotherapy group than in the cytokine group (18 vs 12%). Moreover, the OS rate was also significantly (p < 0.001) higher in the CIK cell-treated group (61 vs 23%). The clinical trials discussed above are summarized in Table 2. Apart from survival and recurrence rates, the quality of life is an important measure to evaluate the benefit of a clinical approach. In some of the above-mentioned studies, the quality of life has been assessed and in all of these studies the physical strength, appetite and sleeping status or the quality of life in general significantly improved after CIK cell therapy [34,44,54]. Liang et al. conducted a study to explicitly evaluate the effect of CIK cell therapy on the quality of life of breast cancer patients who had undergone adjuvant chemotherapy [66]. CIK cell therapy was able to improve the quality of life of breast cancer patients and the patients’ physical and social function was significantly higher in the study group than in the control group.

9.

Conclusion

In the last 2 years since our last review on new adoptive immunotherapy strategies for solid tumors with CIK cells, much effort has been made to evaluate the clinical value of CIK cell therapy and to improve cellular effectivity. CIK cell therapy has been combined with different therapies, resulting in promising clinical responses. Further research is mandatory to support the application of adoptive CIK cell therapy and to integrate this promising therapeutic approach into clinical practice.




10.

Expert opinion

Due to their simplicity in cultivation, their favorable, non-MHC-restricted cytotoxic profile and their potential adaptability to almost any type of tumor, CIK cells are an outstanding approach among adoptive cell therapies. The effort put into NKT/CIK cell research has increased our knowledge of molecular components and mechanisms of CIK/tumor cell interaction. Clinical studies with CIK cells lead to encouraging results -- CIK cell application was assessed as being safe and effective; CIK cell therapy was able to improve the prognosis of cancer patients as it decreased recurrence and increased survival without any harmful side effects. It is assumed that CIK cells might be most effective on residual cancer cells after conventional therapy. Adoptive CIK cell therapy has successfully been applied as adjuvant therapy following surgical resection of the tumor and/or combined with chemotherapy. In the last years, modern therapies have also been combined with CIK cells -- for example, minimally invasive therapies and antiangiogenic drugs. In order to precisely evaluate the effect of the CIK cell immune response in cancer patients, the effect of other therapies, such as chemotherapy, radiotherapy and hormone therapy, should be excluded. However, in routine clinical application and at the latest state of research, it is rather unreasonable to solely treat patients with CIK cells. Moreover, particular control groups are missing in some studies applying multidisciplinary treatments, making definite conclusions imprecise. Unfortunately, most clinical studies include only a relatively low number of patients who are often in advanced or metastatic stage. More clinical trials need to be conducted with larger patient groups, including patients with early tumor stages as well, and involving longer follow-up periods. The CIK cell therapy protocol has to be optimized and standardized. Starting with the cultivation of CIK cells, there are Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Declaration of interest The authors state no conflict of interest and have received no payment for the preparation of this manuscript.

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Affiliation

Clara E Ja¨kel1 & Ingo GH Schmidt-Wolf†2 † Author for correspondence 1 University Hospital Bonn, Center for Integrated Oncology (CIO), Bonn, Germany 2 Professor, University Hospital Bonn, Center for Integrated Oncology (CIO), Sigmund-Freud-Strae 25, 53105 Bonn, Germany Tel: +49 228 287 17050; Fax: +49 228 9080059; E-mail: [email protected]

Adoptive immunotherapy strategies with cytokine-induced killer (CIK) cells in the treatment of hematological malignancies.

Adoptive T-Cell Therapy for Solid Tumors.

New Strategies for the Treatment of Solid Tumors with CAR-T Cells.

New strategies for active immunotherapy with genetically engineered tumor cells.

Mast cells as targets for immunotherapy of solid tumors.

Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy.

Adoptive Immunotherapy For Leukemia With Ex vivo Expanded T Cells.

Reprogramming donor T cells for adoptive immunotherapy.

Targeted immunotherapy for pediatric solid tumors.

Adoptive immunotherapy: New assay for the identification of T cells with optimal avidity.

Adoptive transfer of osteoclast-expanded natural killer cells for immunotherapy targeting cancer stem-like cells in humanized mice.

New strategies for cancer immunotherapy: targeting regulatory T cells.

Modified tumor cells in the immunotherapy of solid mammary tumors.

New cell sources for T cell engineering and adoptive immunotherapy.

Adoptive immunotherapy: a new era for the treatment of cancer.

Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors.

Adoptive Cell Therapy of Melanoma with Cytokine-induced Killer Cells.

Adoptive immunotherapy for myeloid malignancies.

Selection bias: maintaining less-differentiated T cells for adoptive immunotherapy.

An update on allergen immunotherapy and asthma.

Active specific immunotherapy of human solid tumors.

killer T cells for the application to adoptive tumour immunotherapy.

[New perspectives for an adjuvant chemotherapy in solid tumors].

Manipulating immune cells for adoptive immunotherapy of cancer.