The State of Cellular Adoptive Immunotherapy for Neuroblastoma and Other Pediatric Solid Tumors

Research on adult cancer immunotherapy is proceeding at a rapid pace resulting in an impressive success rate exemplified by a few high profile cases. However, this momentum is not readily extended to pediatric immunotherapy, and it is not for lack of trying. Though reasons for the slower advance are not apparent, some issues can be raised. Pediatric cancer patients represent a distinct demographic group whose immune system is inherently different from that of mature adults. Treating pediatric patients with immunotherapy designed for adults may not yield objective clinical responses. Here, we will present an update on adoptive T-cell and natural killer-cell therapies for neuroblastoma and other childhood solid tumors. Additionally, we will delineate key differences between human fetal/neonatal and adult immune systems. We hope this will generate interests leading to the discussion of potential future directions for improving adoptive cancer immunotherapy for children.

ADOPtive t-cell tHerAPY (Act) Adoptive T-cell therapy represents an attractive viable option for the control of solid tumor growth for the following reasons: T cells can systemically home to tumor sites through the entire body and can cross the blood-brain barrier (BBB). By contrast, antibodies such as checkpoint inhibitors cannot effectively cross the BBB and are not consistently or adequately distributed deep inside solid tumors. To date, ACT with CAR T cells is the prevalent type of immunotherapy to treat solid tumors.

Neuroblastoma (Nb)
Neuroblastoma is the most common extracranial solid tumor of childhood and the third most common cause of pediatric cancer death (29)(30)(31)(32). Despite conventional multimodal therapy, patients with high-risk NB have a poor prognosis due to high relapse rate (33). Since 1999, tremendous efforts and funds have been dedicated to discovering and testing various forms of immunotherapy to control refractory/recurrent NB (Table 1). To date the most studied NB-associated antigen (NBAA) identified is the ganglioside GD2 expressed on a subset of NB tumor cells (34). This discovery heralds in the era of GD2-based immunotherapy to treat human NB. The current standard of care for refractory/recurrent NB in human is anti-GD2 therapy, which recognizes and binds GD2 (34)(35)(36)(37)(38)(39). However, because GD2 is also expressed on pain fibers, this therapy has side effects that include severe pain requiring continuous opiate infusions (34). Because of the paucity of identified NBAAs, all CAR clinical trials are GD2-based or variations thereof (38,(40)(41)(42)(43)(44)(45) (Table 1). It is still early to know whether these GD2-dependent CAR trials will yield objective clinical responses. The antigen 4Ig-B7-H3 (CD276), a member of the B7 family of immune regulators such as CD80, CD86, PD-L1, PD-L2 and ICOSL, is expressed in a subset of NB tumors (46)(47)(48)(49). In mouse and human, 4Ig-B7-H3 is expressed on many normal tissues such as spleen, lymph nodes, thymus and fetal liver as well as other tumors. However, its expression is induced on DCs and macrophages by inflammatory cytokines. 4Ig-B7-H3 binds to a yet to be identified cognate receptor induced on activated T and NK cells, and blockade of this interaction results in reduced interferon γ production and loss of cytotoxic activity of these cells. In mice, deficiency or blockade of B7-H3 leads to improved antitumor immunity suggesting that B7-H3 checkpoint may serve as a novel target for immunotherapy against cancers (50). Indeed, a mouse mAb anti-B7-H3 conjugated to iodine 131 (131I-burtomab) has been designed to bind and directly kill NB cells. 131I-burtomab is recently designated a breakthrough drug to treat metastatic NB by the Food and Drug Administration and a clinical trial has been filed (NCT03275402). The immune status of NB tumors treated with 131I-burtomab has not been published. It would be interesting to know whether the antitumor activity of burtomab results from direct killing of tumor cells or activated DCs thus reactivating existing anti-NB immunity. Although no published data are available to show the feasibility of B7-H3 CAR T cells for the treatment of human NB, two patents have been filed for such an invention (US Application No. 14/779,586; US Application No. 61/805,001; PCT Application No. PCT/US2014/031543 and PCT Application No. PCT/US2016/050887; US Application No. 62/216,447). It remains to be determined whether B7-H3 CAR T cells can inhibit NB tumor growth and whether the potential antitumor activity of B7-H3 CAR T cells is due to killing of intratumoral suppressive DCs/macrophages or tumor cells or both.
The anaplastic lymphoma kinase (ALK or CD246) is a receptor protein tyrosine kinase predominantly expressed in the central nervous system (CNS) and peripheral nervous system in mouse and human suggesting its role in normal brain development and function (51). A series of studies show that Alk is frequently mutated (mainly ALK R1275Q and ALK F1174L ) and duplicated in high-risk NB tumors (52)(53)(54)(55)(56)(57)(58). The ALK R1275Q mutation results in a constitutively active kinase suggesting a role for ALK in NB development. However, mice harboring human ALK R1275Q or ALK F1174L alone do not develop aggressive NB irrespective of genetic background (53,57). In the contrary, animals having both MYCN amplification and ALK R1275Q or ALK F1174L mutation succumb to NB at a higher rate (53,57). These findings suggest that mutations in Alk are necessary but not sufficient to drive aggressive NB development. Because ALK is a cell surface kinase, developing CAR T cells targeting ALK has been suggested. Indeed, in a xenogeneic NSG mouse model for NB, human ALK CAR T cells can eradicate ALK-positive tumors; both tumor antigen and receptor density governs the efficacy of these CAR T cells (59). Clinical trials have not been initiated.
Although CAR T-cell therapy is being propelled to the forefront, problems exist that need further investigation. Production of CAR T cells requires the identification of tumor-associated antigen (TAA), generation of an antibody or T-cell receptor (TCR) capable of recognizing the TAA, cloning of genes encoding the antibody or TCR to be introduced into isolated tumorinfiltrating lymphocytes or haploidentical T cells. For most pediatric solid tumors, the identity of TAAs is still unknown and neoantigen load is low thus limiting the use of CAR T cells for this group. In patients with solid tumors for which TAAs have been identified, the use of CAR T cells has proven less effective than in patients with fluid tumors. Recent data are showing a previously unpredicted phenomenon observed in patients treated with CAR T cells: the emergence of tumor cells that have lost expression of the TAA targeted by CAR T cells, undoubtedly due to negative selection imposed by CAR therapy (60)(61)(62). New evidence demonstrates that CAR T cells once in the tumor microenvironment (TME) may suffer from exhaustion caused by suppressor cells including myeloid-derived suppressor cells or CD4 + regulatory T (Treg) cells present in the TME (63)(64)(65). Perhaps a more dangerous issue arisen is the development of cytokine release syndrome (CRS) (66,67) and neurologic toxicity observed in patients undergoing CAR therapy (68).
To circumvent problems posed by CAR T cells, we propose that perhaps the most effective strategy to control solid tumor growth is one that does not require identifying TAAs and corresponding tumor-reactive CD8 + T cells, can enhance effector activity of CD8 + T cells, and can simultaneously eliminate immune suppression within the TME. Our recent studies suggest that such an approach is attainable. We show that by targeting the histone reader Cbx3/HP1γ, we can enhance the tumor killing capacity of effector CD8 + T cells (69,70). As a result, adoptive transfer of Cbx3/HP1γ-deficient CD8 + effector T cells alone into wild type (wt) tumor-bearing mice greatly reduces NB growth. Within the NB TME of Cbx3/HP1γdeficient mice or wt mice treated with Cbx3/HP1γ-deficient CD8 + T cells, we detect an increase of Klrk1/NKG2D + infiltrating CD8 + effector T cells and a decrease in CD4 + Treg cells. PD-1 and Pdl1 expression is not altered in CD8 + T cells or NB tumors, respectively. Moreover, Cbx3/HP1γ-deficient CD8 + T cells appear to have overcome exhaustion. These findings suggest that targeting Cbx3/HP1γ can represent an alternative and rational therapeutic approach to control NB as well as other solid tumors.
Other Pediatric solid tumors (cNs tumors, sarcomas, and Nasopharyngeal sarcomas) As for NB, there is a dearth of identified TAAs available for the formulation of CAR therapy to treat most pediatric solid tumors (71)(72)(73). Tumor immunity against pediatric solid tumors is not completely understood. The human epidermal growth factor receptor 2 (HER2) is expressed on pediatric as well as adult glioblastoma, glioma, and medulloblastoma tumors, and overexpression of HER2 has been associated with poorer prognosis. In animal models, HER2 CAR T cells efficiently cause the regression of CNS tumors. These preclinical studies have paved the way for a few HER2-based CAR clinical trials (74-77) ( Table 2); results of these trials are not yet available. It would be crucial to determine whether HER2-targeted CAR therapy will induce the emergence of HER2-negative tumors as has been shown in animal models and in patients receiving CD19 targeted CAR therapy (60,61) or will cause CRS as in adults (67).
Pediatric nasopharyngeal carcinoma patients with localregional bulky and metastatic disease have a poor prognosis (78). It is a rare tumor that is almost always associated with Epstein-Barr virus (EBV) (78,79), and EBV-specific cytotoxic T lymphocytes (CTLs) can be found in individuals infected with this ubiquitous virus. These findings lead to the design of EBVbased T-cell therapy. In adults, ACT with EBV-specific CTLs is more effective in patients with low disease burden while results for pediatric trials are not available (80-83) ( Table 2).
Prognosis for pediatric patients with recurrent/refractory sarcomas is poor, the survival rate ranges from 10 to 30% (73). For this group of children, few immunotherapy clinical trials are being tested, and past trials using autologous T cells (NCT00001566 and NCT 00001564) have not yielded much information to advance the field ( Table 2). Some studies have shown that the cancer-testes antigen NY-ESO-1 is expressed on a subset of pediatric tumors, which lead to a trial using T cells engineered to express NY-ESO-1-specific TCR (NCT02775292) (84) ( Table 2). HER2 CAR therapy has also been proposed to treat sarcomas (85). CAR T cells targeting glypican-3, a proteoglycan expressed on a small number of solid tumors (86), are being tested in clinical trials to treat pediatric solid tumors (87) (NCT02932956). Data are not yet available for these trials.
The lack of available immunotherapy for pediatric solid tumors may be due to the paucity of identified TAAs, and few basic studies designed to understand tumor immunity during development from infancy to young adulthood in either mouse or human. As a result, most of these trials are based on those that have been designed to treat adult solid tumors.

ADOPtive NK-AND NAtUrAl Killer t (NKt)-cell tHerAPY
Natural killer cells and NKT cells have been shown to play crucial roles in antitumor immunity by directly killing tumor cells or indirectly through antibody-dependent cellular cytotoxicity. Based on results from preclinical studies (88)(89)(90), several clinical trials have been initiated to test the ability of in vitro-activated/expanded or engineered NK cells and NKT cells to control pediatric solid tumors including NB (91,92) (Tables 1 and 2). Results of these trials are not yet available to indicate whether NK-or NKT-cell therapy would be a viable option.
Nonetheless, clinical data have demonstrated that despite the large number of NK cells infused, the antitumor effects of these cells have been modest in adults. NK and NKT cells express a number of inhibitory receptors that bind to MHC class I and other molecules. Additionally, NK and NKT cells are sensitive to various inhibitory molecules within the TME (93). Moreover, we show that the frequency of NK and NKT cells in NB tumors is low, and no differences are detected in tumors from wt or Cbx3/HP1γ-deficient mice yet NB tumor growth is greatly abrogated in Cbx3/HP1γ-deficient mice (70). Our findings imply that NK or NKT cells may not play an important role in controlling NB tumor growth.

DiscUssiON AND FUtUre DirectiONs
In the past 40-50 years, pediatric oncologists have made significant, basic advances toward our understanding of molecular pathways driving the development of tumors in children. This achievement hinges on the belief that cancer pediatric patients represent a distinct demographic group, and the biology of their tumors is fundamentally different from that of adults.
Adult cancer immunotherapy is experiencing a renaissance while that of children is still at its infancy. The momentum that drives adult cancer immunotherapy is built upon decades of basic research designed to understand how an adult immune system responses to tumors developing within an adult host. Thus, there is a need to recognize that pediatric cancer patients represent a distinct demographic group whose immune system is fundamentally different than that of mature adults.
Fetal and adult T cells are distinct populations that arise from different hematopoietic stem cell populations present at different developmental stages (94), and human NK cells follow similar developmental evolution (95,96). Notably, fetal CD4 + T cells are poised to differentiate into CD4 + Treg cells upon allogeneic stimulation. Indeed, human fetus and cord blood (CB) contains an abundance of phenotypically naïve CD25 + CD4 + Treg cells, but functionally mature, capable of suppressing T-and NK-cell proliferation and function (97)(98)(99)(100)(101). Similar suppressive mechanism is observed in the mouse fetus (102). Thus, T-and NK-cell lineages in the developing human or mouse are biased toward immune tolerance mediated by active suppression of early immunity; in some instances, this suppression persists at least until early adulthood (101). The tolerogenic tendency of fetal/neonatal immune system can be attributed to marked differences in response to alloantigens between human fetal and adult DCs. Fetal DCs strongly promote Treg-cell induction and inhibit T-cell tumor-necrosis factor-α production when cultured with alloantigens (103). This may explain why CAR T cells once in the NB TME often suffer from exhaustion. In addition to functional disparities, neonatal and adult immune systems differ quantitatively. Overall, there is a greater number of circulating CD4 + T cells and a lower number of CD8 + T cells in neonates compared to adults (104). Consequently, the ratio of CD4:CD8 is higher in neonates than adults. Together these results suggest that for some children, the persistence of fetal immune suppression, mediated by fetal CD4 + Treg cells, and the lower number of CD8 + T cells may render their immune system incapable of surveilling and eradicating tumor. Therefore in the future, it might be essential to study the effects of persistent fetal immune suppression on tumor development and growth. If the persistence of fetal immune suppression does influence antitumor immunity in pediatric patients, it would be crucial to determine the developmental age at which intervention can be mounted to prevent tumorigenesis and growth without breaking tolerance. Clinically, it might be important to collect data on the immune status of children bearing solid tumors in addition to dissecting their tumor immune environment. Results from these studies might help direct the design of T-and NK-cell therapies that can circumvent suppression and prevent exhaustion induction.
In adult tumors, mutation load appears to correlate with tumor immunity. However, the number of somatic mutations in pediatric solid tumors is low. In the future, it would be necessary to determine mechanisms controlling tumor immunity independent of somatic mutation loads in pediatric patients.
For this demographic group, perhaps the most effective strategy to control solid tumor growth is one that does not require identifying TAAs or neoantigens and their corresponding reactive CD8 + T cells, can enhance effector activity of CD8 + T cells, and can simultaneously eliminate immune suppression within the TME.
We believe these are crucial issues that need to be addressed in order to move the field of pediatric adoptive cancer immunotherapy to the fore. Until there is a will to allow for the funding of such studies and those that are outside traditional belief, children with cancers will continue to be treated as adults, and may not benefit from the cancer immunotherapy renaissance.

AUtHOr cONtribUtiONs
TPL and THT conceived and cowrote this manuscript.

AcKNOWleDGMeNts
We are grateful to all those who have laid significant and important groundwork upon which we build ours. We apologize to those whose studies cannot be cited due to space constraint.