Mol Biol Rep DOI 10.1007/s11033-014-3514-x
Beyond chemotherapy and targeted therapy: adoptive cellular therapy in non-small cell lung cancer Junying Wang • Xueju Wang
Received: 21 December 2013 / Accepted: 19 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Non-small cell lung cancer (NSCLC) is an intractable disease for which effective treatment approaches are urgently needed. The ability to induce antigenspecific immune responses in patients with lung cancer has led to the development of immunotherapy as a novel concept for the treatment of NSCLC. Adoptive cellular therapy (ACT) represents an important advancement in cancer immunotherapy with the utilization of tumor infiltrating lymphocytes, cytokine-induced killer cells, natural killer cells and cd T cells. In this study, we review recent advances in ACT for NSCLC in clinical trials and provide a perspective on the improvement in ACT and potential therapeutic approaches using engineered T cell therapy for NSCLC. Keywords Adoptive immunotherapy Cellular therapy Non-small cell lung cancer
Introduction Lung cancer is the leading cause of cancer-related mortality and one of the most common cancers worldwide [1]. The disease is clinically divided into non-small cell lung
J. Wang (&) Department of Immunology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, Jilin, People’s Republic of China e-mail: [email protected] X. Wang Department of Pathology, The Third Hospital of Jilin University, Changchun 130033, Jilin, People’s Republic of China
cancer (NSCLC) and small cell lung cancer (SCLC). Approximately 85 % of lung cancers are NSCLC, including adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Despite advances in surgical treatment and radiotherapy as well as the introduction of new chemotherapeutic agents and molecular targeted drugs in the past decade, the prognosis of NSCLC remains poor, with an overall 5-year survival rate of 17.1 % [2]. The development of new therapeutic strategies is needed. The host immune system plays a critical role in tumor surveillance and rejection. This observation has led to the development of immunotherapies for various human malignancies. As evidence has emerged confirming the immunogenicity of NSCLC, immune-modulating therapies have emerged as promising avenues for investigation. In this study, we will review several approaches involved in adoptive cellular therapy (ACT), highlighting the opportunities and challenges. We hope this review will provide an overview and contribute to the discovery of the most effective immunotherapy for NSCLC. Is lung cancer immunogenic? Immunogenicity is the ability of an antigen or any substance that might trigger a particular immune reaction. Although lung cancer is not typically regarded as an ‘‘immunogenic’’ malignancy, a growing body of evidence suggests that immune responses to lung tumors might be present, and their magnitude might correlate with patient outcome. Tumor-infiltrating lymphocytes (TILs) represent part of the host immune response against malignancy and might play an important role in cell-mediated immunological destruction of tumors. Kataki et al. [3] demonstrated that 2/3 of tumor stroma inflammatory cells in NSCLC are lymphocytes, and among these, 80 % are T
123
Mol Biol Rep Fig. 1 Adoptive cellular therapy in NSCLC. a NK, CIK, cd T-cells, and TILs expanded ex vivo belong to adoptive immunotherapy, which can kill tumor cells directly. b Engineered T cell therapy. T cells were genetically modified to express a TCR or CAR specific for lung cancer associated antigens
A
B
: tumor associated antigen
cells. Kuo et al. [4] demonstrated a significantly higher number of T cells in NSCLC compared with that in normal tissue. Additionally, the proportion of activated memory T cells was higher in malignant tissues. Evidence is accumulating that high levels of intratumoral TILs are associated with improved recurrence-free survival in NSCLC patients as well as a reduced likelihood of systemic recurrence [5]. A high density of tumor-infiltrating CD4?CD25? regulatory T cells (Tregs) was reported to be associated with the recurrence of resected NSCLC [6]. We and others [7, 8] demonstrated that Tregs found in lung tumors selectively inhibit the host immune response and could contribute to the progression of lung cancer. Adoptive cellular therapy in lung cancer Adoptive cellular therapy for cancer patients relies on the ex vivo generation of tumor-reactive immune cells and administration of the cells in large numbers in the autologous host. Different forms of natural tumor-reactive immune cells have been generated for NSCLC in clinical trials (Fig. 1a), such as TILs, cytokine-induced killer (CIK), cd T cells and natural killer (NK) cells [9]. Immune cells themselves are being increasingly investigated for cancer therapies, with much interest now being focused on T cells, which are key effector cells of the adaptive immune system [10]. An alternative strategy for the rapid generation of tumor specific immune cells is through genetic modification of non-reactive T cells to express a T-cell receptor (TCR) or a chimeric antigen receptor (CAR) (Fig. 1b) that specifically binds to tumor antigens and mediates the antitumor immune response of T cells [11]. Engineered T cell therapy specific for lung cancer associated antigens might provide a novel and hopeful strategy.
123
: TCR T cell
: CAR T cell
TILs therapy Tumor-infiltrating lymphocytes therapy requires the isolation of T cells from fresh cancer specimens and the progressive screening of tumor-reactive T cells ex vivo using high doses of interleukin-2 (IL-2) [12, 13]. Rosenberg and colleagues [14] have clearly demonstrated that adoptive transfer of ex vivo cultured, antigen specific TILs yields durable regression of melanoma. In 1996, TILs therapy was evaluated in 131 NSCLC patients [15]. In this study, TILs were expanded in vitro and infused intravenously into the patients 6–8 weeks after surgery with IL-2 support. This study showed a significant improvement in the overall survival for patients receiving TILs than for those who underwent standard chemoradiotherapy. In particular, the combination of TILs therapy with radiotherapy appeared to be more advantageous than the combination of chemotherapy with radiotherapy in the postoperative treatment of patients with stage III NSCLC. No severe side effects were observed in more than 80 % of the cases. This study suggests that TILs therapy could be useful for NSCLC patients with local advanced disease. Current technical issues with producing tumor-reactive TILs present a formidable barrier to successfully conducting randomized clinical trials. It was reported that only 30–40 % of biopsy specimens yield satisfactory T cell populations, and this protocol of treatment is a very laborand time-intensive procedure, requiring approximately 6 weeks to produce the TILs for infusion [13]. Recently, Dudley et al. [16] established a rapid and simplified method to enrich CD8? ‘‘young’’ TILs for administration as an individualized therapy for advanced melanoma. More recently, Ye et al. [17] elucidated CD137 as a biomarker for naturally occurring tumor-reactive T cells in cancer and developed a rapid, accurate system to isolate and enrich
Mol Biol Rep
rare tumor-reactive TILs populations. These novel techniques might aid in TILs therapy for NSCLC patients. cd T Cell therapy Human cd T cells comprise 1–5 % of the peripheral blood T cells [18]. cd T cells could recognize antigens in a MHCunrestricted manner, and they could bind tumor-expressed ligands, however, not by conventional ab T cells [19]. Most cd T cells lack cell surface expression of CD4 or CD8, in agreement with their MHC-unrestricted recognition of such unconventional antigens. Activated cd T cells display strong cytotoxic effector activity and produce various cytokines, which have recently stimulated great interest in the development of cd T cell-based immunotherapies in NSCLC [19]. Additionally, cd T cells found in TILs were in a more activated state than those in the peripheral blood T cells [20]. Zoledronate is a third-generation amino-bisphosphonate used widely in clinical settings to counteract tumorassociated osteolysis and hypercalcemia [21]. Its use for stimulating the proliferation of cd T cells, with IL-2, which has been approved for clinical use with cancer patients, is expected to be safe for clinical application [22]. After a 2-week expansion in the presence of zoledronate and IL-2, cultured cd T cells could be intravenously administered to patients. The safety of the activated autologous cd T cell–therapy for NSCLC patients was evaluated in a pilot phase I study [23, 24]. In this trial, 15 patients were administered up to 45.1 9 109 (median, 15.7 9 109) cd T cells, and no adverse events were directly related to the cd T cellimmunotherapy. According to the response evaluation criteria in solid tumors (RECIST), there were no objective responses. Six patients had stable disease, whereas the remaining six evaluable patients experienced progressive disease 4 weeks after the sixth transfer. All the patients remained alive during the study period with a median survival of 589 days and median progression-free survival of 126 days. These results demonstrate that adoptive transfer of zoledronate-expanded cd T cells is safe and feasible in patients with NSCLC. To generate better cd T cells for ACT, Kang et al. [25] reported a promising approach for ex vivo expansion of cd T cells using anti-TCR cd antibodies. They showed that antibody-expanded cd T cells contain more effector T cells, which are terminally differentiated cells that display immediate effector functions such as cytokine production and cytotoxicity. The expanded cd T cells showed a significant antitumor effect in vivo, comparable to cisplatinbased chemotherapy. No overt cytotoxic effects against normal cells with kidney, liver and intestine origins were observed. These results suggest that adoptive cd T-cell
therapy will be a safe and effective choice for patients with NSCLC. To further improve the anti-tumor activity of cd T-cell therapy, Hanagiri et al. [26] engineered cd T cells to express the CD8 gene as well as the tumor specific TCR ab genes specific for Kita-Kyushu lung cancer antigen-1 (KKLC-1). The engineered cd T cells showed antigen-specific activity in vitro and in vivo. These results demonstrate that TCR ab-gene transduction into cd T cells is a novel and promising strategy for adoptive immunotherapy. In future clinical applications, combinations of cd T-cell transfer therapy with established surgical, radiotherapy and chemotherapy treatments are expected to improve the survival of lung cancer patients. NK cell therapy NK cells are large granular lymphocytes that are critical effector cells in the early innate immune response to pathogens and cancer [27–29]. These cells account for 10–15 % of the peripheral blood lymphocytes and are phenotypically characterized by the expression of CD56 and an absence of CD3 (i.e., CD56?CD3–). The ability of NK cells to kill tumor cells without the need to recognize a tumor specific Ag provides advantages over T cells and makes them appealing as potential effectors for immunotherapy. Iliopoulou et al. [30] cultured NK cells ex vivo for 20–23 days with interleukin-15 (IL-15) and hydrocortisone (HC) and administered them intravenously between chemotherapy cycles. Fifteen NSCLC patients received 2–4 doses of allogeneic activated NK cells (0.2–29 9 106/kg/ dose, median 4.15 9 106/kg/dose). No local or systemic toxicity was observed. At a median 22-month follow-up, 2 patients with partial response (PR) and six patients with disease stabilization were recorded. The one-year survival rate was 56 % (9/16), and the two-year survival rate was 19 % (3/16). Repetitive infusions of allogeneic, in vitro activated and expanded with IL-15 and HC NK cells, in combination with chemotherapy, are safe and potentially clinically effective. The accumulation of Tregs in the center of lung tumors and in metastatic lymph nodes in combination with a decrease in the NK cell numbers suggests a critical role of Tregs in the formation of an immunosuppressive tumor microenvironment. Lung cancer immunotherapy might be improved by specific Tregs elimination or suppression [31]. NK cells have demonstrated improved clinical outcomes in patients with acute myeloid leukemia when there has been a mismatch between donor NK cells and host MHC molecules [32, 33]. Adoptive transfer of NK cells has been largely unsuccessful for treating patients with solid tumors. Parkhurst et al. [34] conducted a clinical trial to evaluate
123
Mol Biol Rep
the efficacy of NK cells to treat patients with metastatic melanoma or renal cell carcinoma. No clinical responses were observed, even in cases in which high amounts of NK cells were present in the peripheral circulation of patients for at least 1 week post transfer and some patients were treated with adoptively transferred in vitro activated autologous NK cells for several months. Novel strategies to improve NK cell function have been developed [35–37], including the administration of drugs such as thalidomide or imatinib that promote proliferation and activation of NK cells, the blocking of inhibitory signals with anti-killer immunoglobulin-like receptor (KIR) mAbs, the administration of chemotherapy or histone deacetylase inhibitors (HDACi) to upregulate the NKG2D ligands on tumor cells and the administration of proteasome or HDACi to upregulate TNF-related apoptosisinducing ligand (TRAIL) receptor expression in NK cells. In addition, genetic engineering of NK cells to express cytokine transgenes, overexpress activating receptors, or retarget NK cells via chimeric receptors might be effective genetic manipulation approaches to modulate and enhance NK–tumor cell interaction, which shows promise as an effective antitumor therapy [37]. CIK cell therapy CIK cells are a unique population of cytotoxic T lymphocytes with a characteristic CD3?CD56? phenotype [38]. CIK cells are expanded from peripheral blood mononuclear cells (PBMCs) cultured with the timed addition of IFN-c, anti-CD3 Ab, and IL-2 [39], which proliferate rapidly in vitro, with strong antitumor activity and a broad spectrum of targeted tumors [40, 41]. CIK cells regulate and enhance the immune function in cancer patients [41]. Kim et al. [42] showed anti-tumor activity of CIK cells against human NSCLC in vitro and in nude mouse xenograft models. They demonstrated that CIK cells injected intravenously twice per week at dose ranges from 0.3 to 30 9 106 cells per mouse inhibited tumor growth by 32, 67, and 77 % with few side effects. In clinical settings, Li et al. [43] performed a paired study, in which 87 stage I-IV NSCLC patients received autologous CIK-cell immunotherapy in combination with chemotherapy (arm 1) or chemotherapy alone (arm 2). Multivariate analyses indicated that the frequency of CIK-cell immunotherapy was significantly associated with prolonged progression free survival (PFS) (HR 0.91; 95 % CI 0.85–0.98; P = 0.012) and overall survival (OS) (HR 0.83; 95 % CI 0.74–0.93; P = 0.001) in arm 1. This study indicated that CIK-cell treatment could improve the prognosis of NSCLC, and an increased frequency of CIK-cell transfer could result in additional benefits. The antitumor activity of CIK cells is enhanced by activation with dendritic cells (DC), which are
123
the most potent antigen presenting cells [44]. Ren and colleagues [45, 46] demonstrated that the role of DCactivated CIK cells (DC/CIK) in the improvement of chemotherapy in NSCLC treatment is associated with upregulation of the production of cytokines involved in the antitumor effect. Shi et al. [47] showed that DC/CIK treatment prolongs PFS compared with the control group, and no obvious toxicity was reported. Thus, DC/CIK treatment might be a safe and effective method for maintenance therapy for advanced NSCLC. An increasing number of studies on the treatment of patients with cancer using CIK cells have been published worldwide that provide beneficial information for future investigation of CIK cancer therapy. Hontscha et al. [40] established a registry of clinical trials with CIK cells (www.cik-info.org) for various cancers. They collected 16 studies and found that adjuvant immunotherapy with CIK cells might prevent recurrence, improve progression-free survival rates, and improve the quality of life of cancer patients. Combination treatments might further improve the survival rates. More studies are needed to evaluate the efficacy of such treatment strategies for patients with NSCLC. More recently, Shi et al. [48] demonstrated that antiangiogenic therapy enhanced the antitumor activity of adoptively transferred CIK cells in mice, and the synergistic antitumor effect was probably related to the normalized tumor vasculature and reduced hypoxic tumor microenvironment. Thus, combinations of CIK cell therapy with anti-angiogenic therapy are expected to improve the efficiency of ACT in future clinical applications. Potential approaches: engineered T-cell therapy T cells isolated from the blood of patients are reactive against tumors and are generated using genetic engineering, which involves introducing the T cell gene encoding cell surface receptors that are able to recognize tumor associated antigens (TAAs). These receptors could be T-cell receptors (TCRs) derived from responding patients or chimeric antigen receptors (CARs), which are generated using a molecular biology technique [11, 43]. Engineered T-cell therapy, which is actively investigated in clinic, might represent an attractive strategy for NSCLC therapy [11, 43]. Cancer-testis antigens (CTAs) are of particular interest as targets for developing engineered T-cell therapy. CTAs are expressed in a variety of malignant tumors including NSCLC; they are not expressed by normal tissues except in male germline cells and placenta cells, which do not express HLA class I molecules and do not present antigens to T cells [49]. Robbins et al. [50] reported the first adoptive immunotherapy trial to treat solid tumors using T
Mol Biol Rep
cells transduced with TCR directed against NY-ESO-1. They showed that treatment with engineered T cells is effective at mediating tumor regression in patients with metastatic melanoma and synovial cell sarcoma. No ontarget toxicities were seen in this trial because of the lack of NY-ESO-1 expression by normal tissues. Many of the CTAs, such as MAGE, BAGE, GAGE, KK-LC-1 and NYESO-1, are expressed in lung cancers, and additional TCR trials for the treatment of lung cancer patients should be explored in the future. These antigens expanded new possibilities for immunotherapy of lung cancer, and several vaccination trials are underway aimed at inducing a strong CTL response against defined tumor antigens. Immune responses to these CTAs are uncommon in lung cancer patients [51, 52] because of the levels of antigen expression, which are below the threshold for immune recognition. Upregulation of CTA expression by chromatinremodeling agents might enhance the immunogenicity of lung cancer cells, facilitating their eradication by endogenous immune mechanisms or adoptively transferred T cells [53]. CARs are typically composed of an extracellular TAAspecific single-chain antibody variable fragment (scFv) that is linked, via the hinge and transmembrane domains, to an intracellular signaling domain. Recognition of TAAs by CARs is MHC independent, which is a distinct advantage over the TCRs. Folate receptor-alpha (FRA) is a valuable therapeutic target that is highly expressed in a variety of cancers [54], and it exhibits restricted expression in normal adult epithelial tissues; however, FRA attachment at the apical surface of cells situates it away from and out of direct contact with targeted drugs or T cells in circulation. Recent studies [55–57] demonstrated that lung cancers including squamous cell carcinomas and adenocarcinomas strongly express FRA, suggesting that FRA based CAR T cell therapy could potentially be used to treat the majority of lung cancers. The therapeutic success of adoptive therapy with CAR-T cells depends on the appropriate costimulation of TCR-CD3 zeta to induce full T cell activation. These costimulatory cytoplasmic signaling domains are derived from T cell costimulatory molecules. Song et al. [58, 59] demonstrated that anti-FRA CAR outfitted with CD137, CD28 or CD27 costimulatory signaling overcome the issues of engineered T cell persistence and tumor localization that limit the conventional FRA T cell targeting strategy to provide potent antitumor activity in vivo. Recognition of the antigens expressed in normal tissue by engineered T cells presents a major safety concern. ‘‘On-target, off-tumor’’ toxicities have been reported in clinical trials using TCR– and CAR–engineered T cells [60, 61]. Targeting FRA expressed in lung cancer tissue by powerful CAR T cells might induce severe toxicity such as a ‘‘cytokine storm’’ mediated by on-target efficacy
(antitumor effects), which represents a form of off-target toxicity. Introducing suicide gene switches into vectors used to engineer T cells might increase the safety profile and facilitate their clinical application [62]. In recent years, a number of suicide switches have been developed, and inducible caspase 9 (iCasp9) has been shown to efficiently eliminate T cells in patients [63]. Safety might be increased by developing antigen specific RNA engineered-electroporated T cells, in which the toxicity would be expected to abate rapidly because of the transient nature of TCR or CAR expression in T cells [64]. More recently, mRNAengineered T cells could mediate antitumor activity in patients with advanced solid tumors [65].
Conclusions Although new chemotherapeutic agents and several molecularly targeted drugs have been introduced in recent decades, little improvement has been realized in NSCLC survival. Compared with chemotherapy and targeted drug therapy, ACT in NSCLC is a newer concept that is hindered by a lack of long-term clinical experience. Current clinical studies have suggested that adoptive immunotherapy is potent in NSCLC, resulting in an improved clinical outcome and slight toxicity. Combinations of this novel therapy with established surgical treatment, chemotherapy and radiotherapy are expected to improve the efficiency of ACT. Several related factors suggested previously [9] could affect the efficiency of ACT, and large prospective randomized clinical trials to define the roles of ACT in NSCLC are necessary for its improvement as clinical therapy.
Conflict of interest disclosed.
No potential conflicts of interest were
References 1. Siegel R, Naishadham D, Jemal A (2012) Cancer statistics, 2012. CA Cancer J Clin 62(1):10–29 2. Siegel R, DeSantis C, Virgo K, Stein K, Mariotto A, Smith T, Cooper D, Gansler T, Lerro C, Fedewa S, Lin C, Leach C, Cannady RS, Cho H, Scoppa S, Hachey M, Kirch R, Jemal A, Ward E (2012) Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin 62(4):220–241. doi:10.3322/caac.21149 3. Kataki A, Scheid P, Piet M, Marie B, Martinet N, Martinet Y, Vignaud J-M (2002) Tumor infiltrating lymphocytes and macrophages have a potential dual role in lung cancer by supporting both host-defense and tumor progression. J Lab Clin Med 140(5):320–328 4. Kuo SH, Chang DB, Lee YC, Lee YT, Luh KT (1998) Tumourinfiltrating lymphocytes in non-small cell lung cancer are activated T lymphocytes. Respirology 3(1):55–59
123
Mol Biol Rep 5. Al-Shibli KI, Donnem T, Al-Saad S, Persson M, Bremnes RM, Busund L-T (2008) Prognostic effect of epithelial and stromal lymphocyte infiltration in non-small cell lung cancer. Clin Cancer Res 14(16):5220–5227 6. Petersen RP, Campa MJ, Sperlazza J, Conlon D, Joshi MB, Harpole DH Jr, Patz EF Jr (2006) Tumor infiltrating Foxp3? regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients. Cancer 107(12):2866–2872. doi:10. 1002/cncr.22282 7. Woo EY, Yeh H, Chu CS, Schlienger K, Carroll RG, Riley JL, Kaiser LR, June CH (2002) Cutting edge: regulatory T cells from lung cancer patients directly inhibit autologous T cell proliferation. J Immunol 168(9):4272–4276 8. H-y Fu, Li C, Yang W, Gai X-d, Jia T, Lei Y-m, Li Y (2013) FOXP3 and TLR4 protein expression are correlated in non-small cell lung cancer: implications for tumor progression and escape. Acta Histochem 115(2):151–157 9. Zheng Y-W, Li R-M, Zhang X-W, Ren X-B (2013) Current adoptive immunotherapy in non-small cell lung cancer and potential influence of therapy outcome. Cancer Invest 31(3):197–205 10. Medzhitov R, Janeway Jr. CA (1998) Innate immune recognition and control of adaptive immune responses. In: Seminars in immunology. Elsevier, p 351–353 11. Shi H, Liu L, Wang Z (2013) Improving the efficacy and safety of engineered T cell therapy for cancer. Cancer Lett 328(2):191–197 12. Melioli G, Meta M, Semino C, Casartelli G, Pasquetti W, Biassoni R, Ratto G, Catrullo A, Moretta L, Guastella M (1994) Isolation and in vitro expansion of lymphocytes infiltrating nonsmall cell lung carcinoma: functional and molecular characterisation for their use in adoptive immunotherapy. Eur J Cancer 30(1):97–102 13. Dudley ME, Wunderlich JR, Shelton TE, Even J, Rosenberg SA (2003) Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. J Immunother 26(4):332 (Hagerstown, Md: 1997) 14. Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, Citrin DE, Restifo NP, Robbins PF, Wunderlich JR (2011) Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res 17(13):4550–4557 15. Ratto GB, Zino P, Mirabelli S, Minuti P, Aquilina R, Fantino G, Spessa E, Ponte M, Bruzzi P, Melioli G (1996) A randomized trial of adoptive immunotherapy with tumor-infiltrating lymphocytes and interleukin-2 versus standard therapy in the postoperative treatment of resected non-small cell lung carcinoma. Cancer 78(2):244–251 16. Dudley ME, Gross CA, Langhan MM, Garcia MR, Sherry RM, Yang JC, Phan GQ, Kammula US, Hughes MS, Citrin DE (2010) CD8? enriched ‘‘young’’ tumor infiltrating lymphocytes can mediate regression of metastatic melanoma. Clin Cancer Res 16(24):6122–6131 17. Ye Q, Song D, Poussin M, Yamamoto T, Best A, Li C, Coukos G, Powell DJ (2014) CD137 accurately identifies and enriches for naturally-occurring tumor-reactive T cells in tumor. Clin Cancer Res 20:44–55 18. Carding SR, Egan PJ (2002) cd T cells: functional plasticity and heterogeneity. Nat Rev Immunol 2(5):336–345 19. Hayday AC (2000) cd cells: a right time and a right place for a conserved third way of protection. Annu Rev Immunol 18(1):975–1026 20. Yoshida Y, Nakajima J, Wada H, Kakimi K (2011) cd T-cell immunotherapy for lung cancer. Surg Today 41(5):606–611 21. Dhillon S, Lyseng-Williamson KA (2008) Zoledronic acid: a review of its use in the management of bone metastases of malignancy. Drugs 68(4):507–534
123
22. Kondo M, Izumi T, Fujieda N, Kondo A, Morishita T, Matsushita H, Kakimi K (2011) Expansion of human peripheral blood cd T cells using zoledronate. J Vis Exp (55):e3182. doi:10. 3791/3182 23. Sakamoto M, Nakajima J, Murakawa T, Fukami T, Yoshida Y, Murayama T, Takamoto S, Matsushita H, Kakimi K (2011) Adoptive immunotherapy for advanced non-small cell lung cancer using zoledronate-expanded gammadeltaTcells: a phase I clinical study. J Immunother 34(2):202–211. doi:10.1097/CJI. 0b013e318207ecfb 24. Nakajima J, Murakawa T, Fukami T, Goto S, Kaneko T, Yoshida Y, Takamoto S, Kakimi K (2010) A phase I study of adoptive immunotherapy for recurrent non-small-cell lung cancer patients with autologous cd T cells. Eur J Cardiothorac Surg 37(5):1191–1197 25. Kang N, Zhou J, Zhang T, Wang L, Lu F, Cui Y, Cui L, He W (2009) Adoptive immunotherapy of lung cancer with immobilized anti-TCRcd antibody-expanded human cd T Cells in peripheral blood. Cancer Biol Ther 8(16):1540–1549 26. Hanagiri T, Shigematsu Y, Kuroda K, Baba T, Shiota H, Ichiki Y, Nagata Y, Yasuda M, So T, Takenoyama M (2012) Antitumor activity of human cd T cells transducted with CD8 and with T-cell receptors of tumor-specific cytotoxic T lymphocytes. Cancer science 103(8):1414–1419 27. Parham P (2004) Immunology: NK cells lose their inhibition. Sci Signal 305(5685):786 28. Farag SS, Caligiuri MA (2006) Human natural killer cell development and biology. Blood Rev 20(3):123–137 29. Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, Yokoyama WM, Ugolini S (2011) Innate or adaptive immunity? The example of natural killer cells. Science 331(6013):44–49 30. Iliopoulou EG, Kountourakis P, Karamouzis MV, Doufexis D, Ardavanis A, Baxevanis CN, Rigatos G, Papamichail M, Perez SA (2010) A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer. Cancer Immunol Immunother 59(12):1781–1789 31. Schneider T, Kimpfler S, Warth A, Schnabel PA, Dienemann H, Schadendorf D, Hoffmann H, Umansky V (2011) Foxp3? regulatory T cells and natural killer cells distinctly infiltrate primary tumors and draining lymph nodes in pulmonary adenocarcinoma. J Thorac Oncol 6(3):432 32. Miller JS, Soignier Y, Panoskaltsis-Mortari A, McNearney SA, Yun GH, Fautsch SK, McKenna D, Le C, Defor TE, Burns LJ (2005) Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 105(8):3051–3057 33. Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, Posati S, Rogaia D, Frassoni F, Aversa F (2002) Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Sci Signal 295(5562):2097 34. Parkhurst MR, Riley JP, Dudley ME, Rosenberg SA (2011) Adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression. Clin Cancer Res 17(19):6287–6297 35. Ljunggren HG, Malmberg KJ (2007) Prospects for the use of NK cells in immunotherapy of human cancer. Nat Rev Immunol 7(5):329–339 36. Terme M, Ullrich E, Delahaye NF, Chaput N, Zitvogel L (2008) Natural killer cell–directed therapies: moving from unexpected results to successful strategies. Nat Immunol 9(5):486–494 37. Cheng M, Chen Y, Xiao W, Sun R, Tian Z (2013) NK cell-based immunotherapy for malignant diseases. Cell mol immunol 10(3):230–252 38. Jiang J, Wu C, Lu B (2013) Cytokine-induced killer cells promote antitumor immunity. J Transl Med 11(1):83
Mol Biol Rep 39. Lu P-H, Negrin RS (1994) A novel population of expanded human CD3?CD56? cells derived from T cells with potent in vivo antitumor activity in mice with severe combined immunodeficiency. J Immunol 153(4):1687–1696 40. Hontscha C, Borck Y, Zhou H, Messmer D, Schmidt-Wolf IGH (2011) Clinical trials on CIK cells: first report of the international registry on CIK cells (IRCC). J Cancer Res Clin Oncol 137(2):305–310 41. Schmidt-Wolf I, Lefterova P, Mehta B, Fernandez L, Huhn D, Blume K, Weissman I, Negrin R (1993) Phenotypic characterization and identification of effector cells involved in tumor cell recognition of cytokine-induced killer cells. Exp Hematol 21(13):1673 42. Kim HM, Lim J, Park SK, Kang JS, Lee K, Lee CW, Lee KH, Yun MJ, Yang KH, Han G (2007) Antitumor activity of cytokineinduced killer cells against human lung cancer. Int Immunopharmacol 7(13):1802–1807 43. Liu L, Sun M, Wang Z (2012) Adoptive T-cell therapy of B-cell malignancies: conventional and physiological chimeric antigen receptors. Cancer Lett 316(1):1–5 44. Leemhuis T, Wells S, Scheffold C, Edinger M, Negrin RS (2005) A phase I trial of autologous cytokine-induced killer cells for the treatment of relapsed Hodgkin disease and non-Hodgkin lymphoma. Biol Blood Marrow Transplant 11(3):181–187 45. Li H, Wang C, Yu J, Cao S, Wei F, Zhang W, Han Y, Ren X (2009) Dendritic cell-activated cytokine-induced killer cells enhance the anti-tumor effect of chemotherapy on non-small cell lung cancer in patients after surgery. Cytotherapy 11(8):1076–1083 46. Yang L, Ren B, Li H, Yu J, Cao S, Hao X, Ren X (2012) Enhanced antitumor effects of DC-activated CIKs to chemotherapy treatment in a single cohort of advanced non-small-cell lung cancer patients. Cancer Immunol Immunother 62(1):65–73 47. Shi SB, Ma TH, Li CH, Tang XY (2012) Effect of maintenance therapy with dendritic cells: cytokine-induced killer cells in patients with advanced non-small cell lung cancer. Tumori 98(3):314 48. Shi S, Wang R, Chen Y, Song H, Chen L, Huang G (2013) Combining antiangiogenic therapy with adoptive cell immunotherapy exerts better antitumor effects in non-small cell lung cancer models. PLoS ONE 8(6):e65757 49. Chiriva-Internati M, Pandey A, Saba R, Kim M, Saadeh C, Lukman T, Chiaramonte R, Jenkins M, Cobos E, Jumper C (2012) Cancer testis antigens: a novel target in lung cancer. Int Rev Immunol 31(5):321–343 50. Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL (2011) Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 29(7):917–924 51. Stockert E, Ja¨ger E, Chen YT, Scanlan MJ, Gout I, Karbach J, Arand M, Knuth A, Old LJ (1998) A survey of the humoral immune response of cancer patients to a panel of human tumor antigens. J Exp Med 187(8):1349–1354 52. Groeper C, Gambazzi F, Zajac P, Bubendorf L, Adamina M, Rosenthal R, Zerkowski HR, Heberer M, Spagnoli GC (2007) Cancer/testis antigen expression and specific cytotoxic T lymphocyte responses in non small cell lung cancer. Int J Cancer 120(2):337–343
53. Rao M, Chinnasamy N, Hong JA, Zhang Y, Zhang M, Xi S, Liu F, Marquez VE, Morgan RA, Schrump DS (2011) Inhibition of histone lysine methylation enhances cancer-testis antigen expression in lung cancer cells: implications for adoptive immunotherapy of cancer. Cancer Res 71(12):4192–4204 54. Kelemen LE (2006) The role of folate receptor alpha in cancer development, progression and treatment: cause, consequence or innocent bystander? Int J Cancer J Int du Cancer 119(2):243–250. doi:10.1002/ijc.21712 55. O’Shannessy DJ, Yu G, Smale R, Fu Y-S, Singhal S, Thiel RP, Somers EB, Vachani A (2012) Folate receptor alpha expression in lung cancer: diagnostic and prognostic significance. Oncotarget 3(4):414 56. Nunez MI, Behrens C, Woods DM, Lin H, Suraokar M, Kadara H, Hofstetter W, Kalhor N, Lee JJ, Franklin W (2012) High expression of folate receptor alpha in lung cancer correlates with adenocarcinoma histology and EGFR mutation. J Thorac Oncol 7(5):833 57. Cagle PT, Zhai QJ, Murphy L, Low PS (2013) Folate receptor in adenocarcinoma and squamous cell carcinoma of the lung: potential target for folate-linked therapeutic agents. Arch Pathol Lab Med 137(2):241–244 58. Song DG, Ye Q, Carpenito C, Poussin M, Wang LP, Ji C, Figini M, June CH, Coukos G, Powell DJ (2011) In vivo persistence, tumor localization, and antitumor activity of CAR-engineered T cells is enhanced by costimulatory signaling through CD137 (41BB). Cancer Res 71(13):4617–4627 59. Song DG, Ye Q, Poussin M, Harms GM, Figini M, Powell DJ Jr (2012) CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo. Blood 119(3):696–706 60. Parkhurst MR, Yang JC, Langan RC, Dudley ME, Nathan D-AN, Feldman SA, Davis JL, Morgan RA, Merino MJ, Sherry RM (2011) T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 19(3):620–626 61. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA (2010) Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 18(4):843–851 62. Casucci M, Bondanza A (2011) Suicide gene therapy to increase the safety of chimeric antigen receptor-redirected T lymphocytes. J Cancer 2:378 63. Di Stasi A, Tey S-K, Dotti G, Fujita Y, Kennedy-Nasser A, Martinez C, Straathof K, Liu E, Durett AG, Grilley B (2011) Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 365(18):1673–1683 64. Zhao Y, Moon E, Carpenito C, Paulos CM, Liu X, Brennan AL, Chew A, Carroll RG, Scholler J, Levine BL (2010) Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res 70(22):9053–9061 65. Beatty GL, Haas AR, Maus MV, Torigian DA, Soulen MC, Plesa G, Chew A, Zhao Y, Levine BL, Albelda SM (2014) Mesothelinspecific chimeric antigen receptor mrna-engineered T cells induce antitumor activity in solid malignancies. Cancer Immunol Res 2:112–120
123
Beyond chemotherapy and targeted therapy: adoptive cellular therapy in non-small cell lung cancer Junying Wang • Xueju Wang
Received: 21 December 2013 / Accepted: 19 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Non-small cell lung cancer (NSCLC) is an intractable disease for which effective treatment approaches are urgently needed. The ability to induce antigenspecific immune responses in patients with lung cancer has led to the development of immunotherapy as a novel concept for the treatment of NSCLC. Adoptive cellular therapy (ACT) represents an important advancement in cancer immunotherapy with the utilization of tumor infiltrating lymphocytes, cytokine-induced killer cells, natural killer cells and cd T cells. In this study, we review recent advances in ACT for NSCLC in clinical trials and provide a perspective on the improvement in ACT and potential therapeutic approaches using engineered T cell therapy for NSCLC. Keywords Adoptive immunotherapy Cellular therapy Non-small cell lung cancer
Introduction Lung cancer is the leading cause of cancer-related mortality and one of the most common cancers worldwide [1]. The disease is clinically divided into non-small cell lung
J. Wang (&) Department of Immunology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, Jilin, People’s Republic of China e-mail: [email protected] X. Wang Department of Pathology, The Third Hospital of Jilin University, Changchun 130033, Jilin, People’s Republic of China
cancer (NSCLC) and small cell lung cancer (SCLC). Approximately 85 % of lung cancers are NSCLC, including adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Despite advances in surgical treatment and radiotherapy as well as the introduction of new chemotherapeutic agents and molecular targeted drugs in the past decade, the prognosis of NSCLC remains poor, with an overall 5-year survival rate of 17.1 % [2]. The development of new therapeutic strategies is needed. The host immune system plays a critical role in tumor surveillance and rejection. This observation has led to the development of immunotherapies for various human malignancies. As evidence has emerged confirming the immunogenicity of NSCLC, immune-modulating therapies have emerged as promising avenues for investigation. In this study, we will review several approaches involved in adoptive cellular therapy (ACT), highlighting the opportunities and challenges. We hope this review will provide an overview and contribute to the discovery of the most effective immunotherapy for NSCLC. Is lung cancer immunogenic? Immunogenicity is the ability of an antigen or any substance that might trigger a particular immune reaction. Although lung cancer is not typically regarded as an ‘‘immunogenic’’ malignancy, a growing body of evidence suggests that immune responses to lung tumors might be present, and their magnitude might correlate with patient outcome. Tumor-infiltrating lymphocytes (TILs) represent part of the host immune response against malignancy and might play an important role in cell-mediated immunological destruction of tumors. Kataki et al. [3] demonstrated that 2/3 of tumor stroma inflammatory cells in NSCLC are lymphocytes, and among these, 80 % are T
123
Mol Biol Rep Fig. 1 Adoptive cellular therapy in NSCLC. a NK, CIK, cd T-cells, and TILs expanded ex vivo belong to adoptive immunotherapy, which can kill tumor cells directly. b Engineered T cell therapy. T cells were genetically modified to express a TCR or CAR specific for lung cancer associated antigens
A
B
: tumor associated antigen
cells. Kuo et al. [4] demonstrated a significantly higher number of T cells in NSCLC compared with that in normal tissue. Additionally, the proportion of activated memory T cells was higher in malignant tissues. Evidence is accumulating that high levels of intratumoral TILs are associated with improved recurrence-free survival in NSCLC patients as well as a reduced likelihood of systemic recurrence [5]. A high density of tumor-infiltrating CD4?CD25? regulatory T cells (Tregs) was reported to be associated with the recurrence of resected NSCLC [6]. We and others [7, 8] demonstrated that Tregs found in lung tumors selectively inhibit the host immune response and could contribute to the progression of lung cancer. Adoptive cellular therapy in lung cancer Adoptive cellular therapy for cancer patients relies on the ex vivo generation of tumor-reactive immune cells and administration of the cells in large numbers in the autologous host. Different forms of natural tumor-reactive immune cells have been generated for NSCLC in clinical trials (Fig. 1a), such as TILs, cytokine-induced killer (CIK), cd T cells and natural killer (NK) cells [9]. Immune cells themselves are being increasingly investigated for cancer therapies, with much interest now being focused on T cells, which are key effector cells of the adaptive immune system [10]. An alternative strategy for the rapid generation of tumor specific immune cells is through genetic modification of non-reactive T cells to express a T-cell receptor (TCR) or a chimeric antigen receptor (CAR) (Fig. 1b) that specifically binds to tumor antigens and mediates the antitumor immune response of T cells [11]. Engineered T cell therapy specific for lung cancer associated antigens might provide a novel and hopeful strategy.
123
: TCR T cell
: CAR T cell
TILs therapy Tumor-infiltrating lymphocytes therapy requires the isolation of T cells from fresh cancer specimens and the progressive screening of tumor-reactive T cells ex vivo using high doses of interleukin-2 (IL-2) [12, 13]. Rosenberg and colleagues [14] have clearly demonstrated that adoptive transfer of ex vivo cultured, antigen specific TILs yields durable regression of melanoma. In 1996, TILs therapy was evaluated in 131 NSCLC patients [15]. In this study, TILs were expanded in vitro and infused intravenously into the patients 6–8 weeks after surgery with IL-2 support. This study showed a significant improvement in the overall survival for patients receiving TILs than for those who underwent standard chemoradiotherapy. In particular, the combination of TILs therapy with radiotherapy appeared to be more advantageous than the combination of chemotherapy with radiotherapy in the postoperative treatment of patients with stage III NSCLC. No severe side effects were observed in more than 80 % of the cases. This study suggests that TILs therapy could be useful for NSCLC patients with local advanced disease. Current technical issues with producing tumor-reactive TILs present a formidable barrier to successfully conducting randomized clinical trials. It was reported that only 30–40 % of biopsy specimens yield satisfactory T cell populations, and this protocol of treatment is a very laborand time-intensive procedure, requiring approximately 6 weeks to produce the TILs for infusion [13]. Recently, Dudley et al. [16] established a rapid and simplified method to enrich CD8? ‘‘young’’ TILs for administration as an individualized therapy for advanced melanoma. More recently, Ye et al. [17] elucidated CD137 as a biomarker for naturally occurring tumor-reactive T cells in cancer and developed a rapid, accurate system to isolate and enrich
Mol Biol Rep
rare tumor-reactive TILs populations. These novel techniques might aid in TILs therapy for NSCLC patients. cd T Cell therapy Human cd T cells comprise 1–5 % of the peripheral blood T cells [18]. cd T cells could recognize antigens in a MHCunrestricted manner, and they could bind tumor-expressed ligands, however, not by conventional ab T cells [19]. Most cd T cells lack cell surface expression of CD4 or CD8, in agreement with their MHC-unrestricted recognition of such unconventional antigens. Activated cd T cells display strong cytotoxic effector activity and produce various cytokines, which have recently stimulated great interest in the development of cd T cell-based immunotherapies in NSCLC [19]. Additionally, cd T cells found in TILs were in a more activated state than those in the peripheral blood T cells [20]. Zoledronate is a third-generation amino-bisphosphonate used widely in clinical settings to counteract tumorassociated osteolysis and hypercalcemia [21]. Its use for stimulating the proliferation of cd T cells, with IL-2, which has been approved for clinical use with cancer patients, is expected to be safe for clinical application [22]. After a 2-week expansion in the presence of zoledronate and IL-2, cultured cd T cells could be intravenously administered to patients. The safety of the activated autologous cd T cell–therapy for NSCLC patients was evaluated in a pilot phase I study [23, 24]. In this trial, 15 patients were administered up to 45.1 9 109 (median, 15.7 9 109) cd T cells, and no adverse events were directly related to the cd T cellimmunotherapy. According to the response evaluation criteria in solid tumors (RECIST), there were no objective responses. Six patients had stable disease, whereas the remaining six evaluable patients experienced progressive disease 4 weeks after the sixth transfer. All the patients remained alive during the study period with a median survival of 589 days and median progression-free survival of 126 days. These results demonstrate that adoptive transfer of zoledronate-expanded cd T cells is safe and feasible in patients with NSCLC. To generate better cd T cells for ACT, Kang et al. [25] reported a promising approach for ex vivo expansion of cd T cells using anti-TCR cd antibodies. They showed that antibody-expanded cd T cells contain more effector T cells, which are terminally differentiated cells that display immediate effector functions such as cytokine production and cytotoxicity. The expanded cd T cells showed a significant antitumor effect in vivo, comparable to cisplatinbased chemotherapy. No overt cytotoxic effects against normal cells with kidney, liver and intestine origins were observed. These results suggest that adoptive cd T-cell
therapy will be a safe and effective choice for patients with NSCLC. To further improve the anti-tumor activity of cd T-cell therapy, Hanagiri et al. [26] engineered cd T cells to express the CD8 gene as well as the tumor specific TCR ab genes specific for Kita-Kyushu lung cancer antigen-1 (KKLC-1). The engineered cd T cells showed antigen-specific activity in vitro and in vivo. These results demonstrate that TCR ab-gene transduction into cd T cells is a novel and promising strategy for adoptive immunotherapy. In future clinical applications, combinations of cd T-cell transfer therapy with established surgical, radiotherapy and chemotherapy treatments are expected to improve the survival of lung cancer patients. NK cell therapy NK cells are large granular lymphocytes that are critical effector cells in the early innate immune response to pathogens and cancer [27–29]. These cells account for 10–15 % of the peripheral blood lymphocytes and are phenotypically characterized by the expression of CD56 and an absence of CD3 (i.e., CD56?CD3–). The ability of NK cells to kill tumor cells without the need to recognize a tumor specific Ag provides advantages over T cells and makes them appealing as potential effectors for immunotherapy. Iliopoulou et al. [30] cultured NK cells ex vivo for 20–23 days with interleukin-15 (IL-15) and hydrocortisone (HC) and administered them intravenously between chemotherapy cycles. Fifteen NSCLC patients received 2–4 doses of allogeneic activated NK cells (0.2–29 9 106/kg/ dose, median 4.15 9 106/kg/dose). No local or systemic toxicity was observed. At a median 22-month follow-up, 2 patients with partial response (PR) and six patients with disease stabilization were recorded. The one-year survival rate was 56 % (9/16), and the two-year survival rate was 19 % (3/16). Repetitive infusions of allogeneic, in vitro activated and expanded with IL-15 and HC NK cells, in combination with chemotherapy, are safe and potentially clinically effective. The accumulation of Tregs in the center of lung tumors and in metastatic lymph nodes in combination with a decrease in the NK cell numbers suggests a critical role of Tregs in the formation of an immunosuppressive tumor microenvironment. Lung cancer immunotherapy might be improved by specific Tregs elimination or suppression [31]. NK cells have demonstrated improved clinical outcomes in patients with acute myeloid leukemia when there has been a mismatch between donor NK cells and host MHC molecules [32, 33]. Adoptive transfer of NK cells has been largely unsuccessful for treating patients with solid tumors. Parkhurst et al. [34] conducted a clinical trial to evaluate
123
Mol Biol Rep
the efficacy of NK cells to treat patients with metastatic melanoma or renal cell carcinoma. No clinical responses were observed, even in cases in which high amounts of NK cells were present in the peripheral circulation of patients for at least 1 week post transfer and some patients were treated with adoptively transferred in vitro activated autologous NK cells for several months. Novel strategies to improve NK cell function have been developed [35–37], including the administration of drugs such as thalidomide or imatinib that promote proliferation and activation of NK cells, the blocking of inhibitory signals with anti-killer immunoglobulin-like receptor (KIR) mAbs, the administration of chemotherapy or histone deacetylase inhibitors (HDACi) to upregulate the NKG2D ligands on tumor cells and the administration of proteasome or HDACi to upregulate TNF-related apoptosisinducing ligand (TRAIL) receptor expression in NK cells. In addition, genetic engineering of NK cells to express cytokine transgenes, overexpress activating receptors, or retarget NK cells via chimeric receptors might be effective genetic manipulation approaches to modulate and enhance NK–tumor cell interaction, which shows promise as an effective antitumor therapy [37]. CIK cell therapy CIK cells are a unique population of cytotoxic T lymphocytes with a characteristic CD3?CD56? phenotype [38]. CIK cells are expanded from peripheral blood mononuclear cells (PBMCs) cultured with the timed addition of IFN-c, anti-CD3 Ab, and IL-2 [39], which proliferate rapidly in vitro, with strong antitumor activity and a broad spectrum of targeted tumors [40, 41]. CIK cells regulate and enhance the immune function in cancer patients [41]. Kim et al. [42] showed anti-tumor activity of CIK cells against human NSCLC in vitro and in nude mouse xenograft models. They demonstrated that CIK cells injected intravenously twice per week at dose ranges from 0.3 to 30 9 106 cells per mouse inhibited tumor growth by 32, 67, and 77 % with few side effects. In clinical settings, Li et al. [43] performed a paired study, in which 87 stage I-IV NSCLC patients received autologous CIK-cell immunotherapy in combination with chemotherapy (arm 1) or chemotherapy alone (arm 2). Multivariate analyses indicated that the frequency of CIK-cell immunotherapy was significantly associated with prolonged progression free survival (PFS) (HR 0.91; 95 % CI 0.85–0.98; P = 0.012) and overall survival (OS) (HR 0.83; 95 % CI 0.74–0.93; P = 0.001) in arm 1. This study indicated that CIK-cell treatment could improve the prognosis of NSCLC, and an increased frequency of CIK-cell transfer could result in additional benefits. The antitumor activity of CIK cells is enhanced by activation with dendritic cells (DC), which are
123
the most potent antigen presenting cells [44]. Ren and colleagues [45, 46] demonstrated that the role of DCactivated CIK cells (DC/CIK) in the improvement of chemotherapy in NSCLC treatment is associated with upregulation of the production of cytokines involved in the antitumor effect. Shi et al. [47] showed that DC/CIK treatment prolongs PFS compared with the control group, and no obvious toxicity was reported. Thus, DC/CIK treatment might be a safe and effective method for maintenance therapy for advanced NSCLC. An increasing number of studies on the treatment of patients with cancer using CIK cells have been published worldwide that provide beneficial information for future investigation of CIK cancer therapy. Hontscha et al. [40] established a registry of clinical trials with CIK cells (www.cik-info.org) for various cancers. They collected 16 studies and found that adjuvant immunotherapy with CIK cells might prevent recurrence, improve progression-free survival rates, and improve the quality of life of cancer patients. Combination treatments might further improve the survival rates. More studies are needed to evaluate the efficacy of such treatment strategies for patients with NSCLC. More recently, Shi et al. [48] demonstrated that antiangiogenic therapy enhanced the antitumor activity of adoptively transferred CIK cells in mice, and the synergistic antitumor effect was probably related to the normalized tumor vasculature and reduced hypoxic tumor microenvironment. Thus, combinations of CIK cell therapy with anti-angiogenic therapy are expected to improve the efficiency of ACT in future clinical applications. Potential approaches: engineered T-cell therapy T cells isolated from the blood of patients are reactive against tumors and are generated using genetic engineering, which involves introducing the T cell gene encoding cell surface receptors that are able to recognize tumor associated antigens (TAAs). These receptors could be T-cell receptors (TCRs) derived from responding patients or chimeric antigen receptors (CARs), which are generated using a molecular biology technique [11, 43]. Engineered T-cell therapy, which is actively investigated in clinic, might represent an attractive strategy for NSCLC therapy [11, 43]. Cancer-testis antigens (CTAs) are of particular interest as targets for developing engineered T-cell therapy. CTAs are expressed in a variety of malignant tumors including NSCLC; they are not expressed by normal tissues except in male germline cells and placenta cells, which do not express HLA class I molecules and do not present antigens to T cells [49]. Robbins et al. [50] reported the first adoptive immunotherapy trial to treat solid tumors using T
Mol Biol Rep
cells transduced with TCR directed against NY-ESO-1. They showed that treatment with engineered T cells is effective at mediating tumor regression in patients with metastatic melanoma and synovial cell sarcoma. No ontarget toxicities were seen in this trial because of the lack of NY-ESO-1 expression by normal tissues. Many of the CTAs, such as MAGE, BAGE, GAGE, KK-LC-1 and NYESO-1, are expressed in lung cancers, and additional TCR trials for the treatment of lung cancer patients should be explored in the future. These antigens expanded new possibilities for immunotherapy of lung cancer, and several vaccination trials are underway aimed at inducing a strong CTL response against defined tumor antigens. Immune responses to these CTAs are uncommon in lung cancer patients [51, 52] because of the levels of antigen expression, which are below the threshold for immune recognition. Upregulation of CTA expression by chromatinremodeling agents might enhance the immunogenicity of lung cancer cells, facilitating their eradication by endogenous immune mechanisms or adoptively transferred T cells [53]. CARs are typically composed of an extracellular TAAspecific single-chain antibody variable fragment (scFv) that is linked, via the hinge and transmembrane domains, to an intracellular signaling domain. Recognition of TAAs by CARs is MHC independent, which is a distinct advantage over the TCRs. Folate receptor-alpha (FRA) is a valuable therapeutic target that is highly expressed in a variety of cancers [54], and it exhibits restricted expression in normal adult epithelial tissues; however, FRA attachment at the apical surface of cells situates it away from and out of direct contact with targeted drugs or T cells in circulation. Recent studies [55–57] demonstrated that lung cancers including squamous cell carcinomas and adenocarcinomas strongly express FRA, suggesting that FRA based CAR T cell therapy could potentially be used to treat the majority of lung cancers. The therapeutic success of adoptive therapy with CAR-T cells depends on the appropriate costimulation of TCR-CD3 zeta to induce full T cell activation. These costimulatory cytoplasmic signaling domains are derived from T cell costimulatory molecules. Song et al. [58, 59] demonstrated that anti-FRA CAR outfitted with CD137, CD28 or CD27 costimulatory signaling overcome the issues of engineered T cell persistence and tumor localization that limit the conventional FRA T cell targeting strategy to provide potent antitumor activity in vivo. Recognition of the antigens expressed in normal tissue by engineered T cells presents a major safety concern. ‘‘On-target, off-tumor’’ toxicities have been reported in clinical trials using TCR– and CAR–engineered T cells [60, 61]. Targeting FRA expressed in lung cancer tissue by powerful CAR T cells might induce severe toxicity such as a ‘‘cytokine storm’’ mediated by on-target efficacy
(antitumor effects), which represents a form of off-target toxicity. Introducing suicide gene switches into vectors used to engineer T cells might increase the safety profile and facilitate their clinical application [62]. In recent years, a number of suicide switches have been developed, and inducible caspase 9 (iCasp9) has been shown to efficiently eliminate T cells in patients [63]. Safety might be increased by developing antigen specific RNA engineered-electroporated T cells, in which the toxicity would be expected to abate rapidly because of the transient nature of TCR or CAR expression in T cells [64]. More recently, mRNAengineered T cells could mediate antitumor activity in patients with advanced solid tumors [65].
Conclusions Although new chemotherapeutic agents and several molecularly targeted drugs have been introduced in recent decades, little improvement has been realized in NSCLC survival. Compared with chemotherapy and targeted drug therapy, ACT in NSCLC is a newer concept that is hindered by a lack of long-term clinical experience. Current clinical studies have suggested that adoptive immunotherapy is potent in NSCLC, resulting in an improved clinical outcome and slight toxicity. Combinations of this novel therapy with established surgical treatment, chemotherapy and radiotherapy are expected to improve the efficiency of ACT. Several related factors suggested previously [9] could affect the efficiency of ACT, and large prospective randomized clinical trials to define the roles of ACT in NSCLC are necessary for its improvement as clinical therapy.
Conflict of interest disclosed.
No potential conflicts of interest were
References 1. Siegel R, Naishadham D, Jemal A (2012) Cancer statistics, 2012. CA Cancer J Clin 62(1):10–29 2. Siegel R, DeSantis C, Virgo K, Stein K, Mariotto A, Smith T, Cooper D, Gansler T, Lerro C, Fedewa S, Lin C, Leach C, Cannady RS, Cho H, Scoppa S, Hachey M, Kirch R, Jemal A, Ward E (2012) Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin 62(4):220–241. doi:10.3322/caac.21149 3. Kataki A, Scheid P, Piet M, Marie B, Martinet N, Martinet Y, Vignaud J-M (2002) Tumor infiltrating lymphocytes and macrophages have a potential dual role in lung cancer by supporting both host-defense and tumor progression. J Lab Clin Med 140(5):320–328 4. Kuo SH, Chang DB, Lee YC, Lee YT, Luh KT (1998) Tumourinfiltrating lymphocytes in non-small cell lung cancer are activated T lymphocytes. Respirology 3(1):55–59
123
Mol Biol Rep 5. Al-Shibli KI, Donnem T, Al-Saad S, Persson M, Bremnes RM, Busund L-T (2008) Prognostic effect of epithelial and stromal lymphocyte infiltration in non-small cell lung cancer. Clin Cancer Res 14(16):5220–5227 6. Petersen RP, Campa MJ, Sperlazza J, Conlon D, Joshi MB, Harpole DH Jr, Patz EF Jr (2006) Tumor infiltrating Foxp3? regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients. Cancer 107(12):2866–2872. doi:10. 1002/cncr.22282 7. Woo EY, Yeh H, Chu CS, Schlienger K, Carroll RG, Riley JL, Kaiser LR, June CH (2002) Cutting edge: regulatory T cells from lung cancer patients directly inhibit autologous T cell proliferation. J Immunol 168(9):4272–4276 8. H-y Fu, Li C, Yang W, Gai X-d, Jia T, Lei Y-m, Li Y (2013) FOXP3 and TLR4 protein expression are correlated in non-small cell lung cancer: implications for tumor progression and escape. Acta Histochem 115(2):151–157 9. Zheng Y-W, Li R-M, Zhang X-W, Ren X-B (2013) Current adoptive immunotherapy in non-small cell lung cancer and potential influence of therapy outcome. Cancer Invest 31(3):197–205 10. Medzhitov R, Janeway Jr. CA (1998) Innate immune recognition and control of adaptive immune responses. In: Seminars in immunology. Elsevier, p 351–353 11. Shi H, Liu L, Wang Z (2013) Improving the efficacy and safety of engineered T cell therapy for cancer. Cancer Lett 328(2):191–197 12. Melioli G, Meta M, Semino C, Casartelli G, Pasquetti W, Biassoni R, Ratto G, Catrullo A, Moretta L, Guastella M (1994) Isolation and in vitro expansion of lymphocytes infiltrating nonsmall cell lung carcinoma: functional and molecular characterisation for their use in adoptive immunotherapy. Eur J Cancer 30(1):97–102 13. Dudley ME, Wunderlich JR, Shelton TE, Even J, Rosenberg SA (2003) Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. J Immunother 26(4):332 (Hagerstown, Md: 1997) 14. Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, Citrin DE, Restifo NP, Robbins PF, Wunderlich JR (2011) Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res 17(13):4550–4557 15. Ratto GB, Zino P, Mirabelli S, Minuti P, Aquilina R, Fantino G, Spessa E, Ponte M, Bruzzi P, Melioli G (1996) A randomized trial of adoptive immunotherapy with tumor-infiltrating lymphocytes and interleukin-2 versus standard therapy in the postoperative treatment of resected non-small cell lung carcinoma. Cancer 78(2):244–251 16. Dudley ME, Gross CA, Langhan MM, Garcia MR, Sherry RM, Yang JC, Phan GQ, Kammula US, Hughes MS, Citrin DE (2010) CD8? enriched ‘‘young’’ tumor infiltrating lymphocytes can mediate regression of metastatic melanoma. Clin Cancer Res 16(24):6122–6131 17. Ye Q, Song D, Poussin M, Yamamoto T, Best A, Li C, Coukos G, Powell DJ (2014) CD137 accurately identifies and enriches for naturally-occurring tumor-reactive T cells in tumor. Clin Cancer Res 20:44–55 18. Carding SR, Egan PJ (2002) cd T cells: functional plasticity and heterogeneity. Nat Rev Immunol 2(5):336–345 19. Hayday AC (2000) cd cells: a right time and a right place for a conserved third way of protection. Annu Rev Immunol 18(1):975–1026 20. Yoshida Y, Nakajima J, Wada H, Kakimi K (2011) cd T-cell immunotherapy for lung cancer. Surg Today 41(5):606–611 21. Dhillon S, Lyseng-Williamson KA (2008) Zoledronic acid: a review of its use in the management of bone metastases of malignancy. Drugs 68(4):507–534
123
22. Kondo M, Izumi T, Fujieda N, Kondo A, Morishita T, Matsushita H, Kakimi K (2011) Expansion of human peripheral blood cd T cells using zoledronate. J Vis Exp (55):e3182. doi:10. 3791/3182 23. Sakamoto M, Nakajima J, Murakawa T, Fukami T, Yoshida Y, Murayama T, Takamoto S, Matsushita H, Kakimi K (2011) Adoptive immunotherapy for advanced non-small cell lung cancer using zoledronate-expanded gammadeltaTcells: a phase I clinical study. J Immunother 34(2):202–211. doi:10.1097/CJI. 0b013e318207ecfb 24. Nakajima J, Murakawa T, Fukami T, Goto S, Kaneko T, Yoshida Y, Takamoto S, Kakimi K (2010) A phase I study of adoptive immunotherapy for recurrent non-small-cell lung cancer patients with autologous cd T cells. Eur J Cardiothorac Surg 37(5):1191–1197 25. Kang N, Zhou J, Zhang T, Wang L, Lu F, Cui Y, Cui L, He W (2009) Adoptive immunotherapy of lung cancer with immobilized anti-TCRcd antibody-expanded human cd T Cells in peripheral blood. Cancer Biol Ther 8(16):1540–1549 26. Hanagiri T, Shigematsu Y, Kuroda K, Baba T, Shiota H, Ichiki Y, Nagata Y, Yasuda M, So T, Takenoyama M (2012) Antitumor activity of human cd T cells transducted with CD8 and with T-cell receptors of tumor-specific cytotoxic T lymphocytes. Cancer science 103(8):1414–1419 27. Parham P (2004) Immunology: NK cells lose their inhibition. Sci Signal 305(5685):786 28. Farag SS, Caligiuri MA (2006) Human natural killer cell development and biology. Blood Rev 20(3):123–137 29. Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, Yokoyama WM, Ugolini S (2011) Innate or adaptive immunity? The example of natural killer cells. Science 331(6013):44–49 30. Iliopoulou EG, Kountourakis P, Karamouzis MV, Doufexis D, Ardavanis A, Baxevanis CN, Rigatos G, Papamichail M, Perez SA (2010) A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer. Cancer Immunol Immunother 59(12):1781–1789 31. Schneider T, Kimpfler S, Warth A, Schnabel PA, Dienemann H, Schadendorf D, Hoffmann H, Umansky V (2011) Foxp3? regulatory T cells and natural killer cells distinctly infiltrate primary tumors and draining lymph nodes in pulmonary adenocarcinoma. J Thorac Oncol 6(3):432 32. Miller JS, Soignier Y, Panoskaltsis-Mortari A, McNearney SA, Yun GH, Fautsch SK, McKenna D, Le C, Defor TE, Burns LJ (2005) Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 105(8):3051–3057 33. Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, Posati S, Rogaia D, Frassoni F, Aversa F (2002) Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Sci Signal 295(5562):2097 34. Parkhurst MR, Riley JP, Dudley ME, Rosenberg SA (2011) Adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression. Clin Cancer Res 17(19):6287–6297 35. Ljunggren HG, Malmberg KJ (2007) Prospects for the use of NK cells in immunotherapy of human cancer. Nat Rev Immunol 7(5):329–339 36. Terme M, Ullrich E, Delahaye NF, Chaput N, Zitvogel L (2008) Natural killer cell–directed therapies: moving from unexpected results to successful strategies. Nat Immunol 9(5):486–494 37. Cheng M, Chen Y, Xiao W, Sun R, Tian Z (2013) NK cell-based immunotherapy for malignant diseases. Cell mol immunol 10(3):230–252 38. Jiang J, Wu C, Lu B (2013) Cytokine-induced killer cells promote antitumor immunity. J Transl Med 11(1):83
Mol Biol Rep 39. Lu P-H, Negrin RS (1994) A novel population of expanded human CD3?CD56? cells derived from T cells with potent in vivo antitumor activity in mice with severe combined immunodeficiency. J Immunol 153(4):1687–1696 40. Hontscha C, Borck Y, Zhou H, Messmer D, Schmidt-Wolf IGH (2011) Clinical trials on CIK cells: first report of the international registry on CIK cells (IRCC). J Cancer Res Clin Oncol 137(2):305–310 41. Schmidt-Wolf I, Lefterova P, Mehta B, Fernandez L, Huhn D, Blume K, Weissman I, Negrin R (1993) Phenotypic characterization and identification of effector cells involved in tumor cell recognition of cytokine-induced killer cells. Exp Hematol 21(13):1673 42. Kim HM, Lim J, Park SK, Kang JS, Lee K, Lee CW, Lee KH, Yun MJ, Yang KH, Han G (2007) Antitumor activity of cytokineinduced killer cells against human lung cancer. Int Immunopharmacol 7(13):1802–1807 43. Liu L, Sun M, Wang Z (2012) Adoptive T-cell therapy of B-cell malignancies: conventional and physiological chimeric antigen receptors. Cancer Lett 316(1):1–5 44. Leemhuis T, Wells S, Scheffold C, Edinger M, Negrin RS (2005) A phase I trial of autologous cytokine-induced killer cells for the treatment of relapsed Hodgkin disease and non-Hodgkin lymphoma. Biol Blood Marrow Transplant 11(3):181–187 45. Li H, Wang C, Yu J, Cao S, Wei F, Zhang W, Han Y, Ren X (2009) Dendritic cell-activated cytokine-induced killer cells enhance the anti-tumor effect of chemotherapy on non-small cell lung cancer in patients after surgery. Cytotherapy 11(8):1076–1083 46. Yang L, Ren B, Li H, Yu J, Cao S, Hao X, Ren X (2012) Enhanced antitumor effects of DC-activated CIKs to chemotherapy treatment in a single cohort of advanced non-small-cell lung cancer patients. Cancer Immunol Immunother 62(1):65–73 47. Shi SB, Ma TH, Li CH, Tang XY (2012) Effect of maintenance therapy with dendritic cells: cytokine-induced killer cells in patients with advanced non-small cell lung cancer. Tumori 98(3):314 48. Shi S, Wang R, Chen Y, Song H, Chen L, Huang G (2013) Combining antiangiogenic therapy with adoptive cell immunotherapy exerts better antitumor effects in non-small cell lung cancer models. PLoS ONE 8(6):e65757 49. Chiriva-Internati M, Pandey A, Saba R, Kim M, Saadeh C, Lukman T, Chiaramonte R, Jenkins M, Cobos E, Jumper C (2012) Cancer testis antigens: a novel target in lung cancer. Int Rev Immunol 31(5):321–343 50. Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL (2011) Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 29(7):917–924 51. Stockert E, Ja¨ger E, Chen YT, Scanlan MJ, Gout I, Karbach J, Arand M, Knuth A, Old LJ (1998) A survey of the humoral immune response of cancer patients to a panel of human tumor antigens. J Exp Med 187(8):1349–1354 52. Groeper C, Gambazzi F, Zajac P, Bubendorf L, Adamina M, Rosenthal R, Zerkowski HR, Heberer M, Spagnoli GC (2007) Cancer/testis antigen expression and specific cytotoxic T lymphocyte responses in non small cell lung cancer. Int J Cancer 120(2):337–343
53. Rao M, Chinnasamy N, Hong JA, Zhang Y, Zhang M, Xi S, Liu F, Marquez VE, Morgan RA, Schrump DS (2011) Inhibition of histone lysine methylation enhances cancer-testis antigen expression in lung cancer cells: implications for adoptive immunotherapy of cancer. Cancer Res 71(12):4192–4204 54. Kelemen LE (2006) The role of folate receptor alpha in cancer development, progression and treatment: cause, consequence or innocent bystander? Int J Cancer J Int du Cancer 119(2):243–250. doi:10.1002/ijc.21712 55. O’Shannessy DJ, Yu G, Smale R, Fu Y-S, Singhal S, Thiel RP, Somers EB, Vachani A (2012) Folate receptor alpha expression in lung cancer: diagnostic and prognostic significance. Oncotarget 3(4):414 56. Nunez MI, Behrens C, Woods DM, Lin H, Suraokar M, Kadara H, Hofstetter W, Kalhor N, Lee JJ, Franklin W (2012) High expression of folate receptor alpha in lung cancer correlates with adenocarcinoma histology and EGFR mutation. J Thorac Oncol 7(5):833 57. Cagle PT, Zhai QJ, Murphy L, Low PS (2013) Folate receptor in adenocarcinoma and squamous cell carcinoma of the lung: potential target for folate-linked therapeutic agents. Arch Pathol Lab Med 137(2):241–244 58. Song DG, Ye Q, Carpenito C, Poussin M, Wang LP, Ji C, Figini M, June CH, Coukos G, Powell DJ (2011) In vivo persistence, tumor localization, and antitumor activity of CAR-engineered T cells is enhanced by costimulatory signaling through CD137 (41BB). Cancer Res 71(13):4617–4627 59. Song DG, Ye Q, Poussin M, Harms GM, Figini M, Powell DJ Jr (2012) CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo. Blood 119(3):696–706 60. Parkhurst MR, Yang JC, Langan RC, Dudley ME, Nathan D-AN, Feldman SA, Davis JL, Morgan RA, Merino MJ, Sherry RM (2011) T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 19(3):620–626 61. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA (2010) Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 18(4):843–851 62. Casucci M, Bondanza A (2011) Suicide gene therapy to increase the safety of chimeric antigen receptor-redirected T lymphocytes. J Cancer 2:378 63. Di Stasi A, Tey S-K, Dotti G, Fujita Y, Kennedy-Nasser A, Martinez C, Straathof K, Liu E, Durett AG, Grilley B (2011) Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 365(18):1673–1683 64. Zhao Y, Moon E, Carpenito C, Paulos CM, Liu X, Brennan AL, Chew A, Carroll RG, Scholler J, Levine BL (2010) Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res 70(22):9053–9061 65. Beatty GL, Haas AR, Maus MV, Torigian DA, Soulen MC, Plesa G, Chew A, Zhao Y, Levine BL, Albelda SM (2014) Mesothelinspecific chimeric antigen receptor mrna-engineered T cells induce antitumor activity in solid malignancies. Cancer Immunol Res 2:112–120
123
