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CONTENTSREVIEW: Immunomodulatory effects of radiation: what is next for cancer therapy? Future Oncology Vol. 12 Issue 2

REVIEW: Immunotherapy in prostate cancer: challenges and opportunities Immunotherapy Vol. 8 Issue 1

REVIEW: Vaccinia virus, a promising new therapeutic agent for pancreatic cancer Immunotherapy Vol. 7 Issue 12

REVIEW: Checkpoint inhibitors in bladder and renal cancers: results and perspectives Immunotherapy Vol. 7 Issue 12

REVIEW: Emerging targets in cancer immunotherapy: beyond CTLA-4 and PD-1 Immunotherapy Vol. 7 Issue 11

REVIEW: Emerging immunotherapy in pediatric lymphoma Future Oncology Vol. 12 Issue 2

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REVIEW

Immunomodulatory effects of radiation: what is next for cancer therapy?

Anita Kumari1, Samantha S Simon1, Tomika D Moody1 & Charlie Garnett-Benson*,1

1Department of Biology, Georgia State University, 161 Jesse Hill Jr Dr, Atlanta, GA, USA

*Author for correspondence: Tel.: +1 404 413 5441; [email protected]

Despite its former reputation as being immunosuppressive, it has become evident that radiation therapy can enhance antitumor immune responses. This quality can be harnessed by utilizing radiation as an adjuvant to cancer immunotherapies. Most studies combine the standard radiation dose and regimens indicated for the given disease state, with novel cancer immunotherapies. It has become apparent that low-dose radiation, as well as doses within the hypofractionated range, can modulate tumor cells making them better targets for immune cell reactivity. Herein, we describe the range of phenotypic changes induced in tumor cells by radiation, and explore the diverse mechanisms of immunogenic modulation reported at these doses. We also review the impact of these doses on the immune cell function of cytotoxic cells in vivo and in vitro.

First draft submitted: 17 August 2015; Accepted for publication: 26 October 2015; Published online: 1 December 2015

KEYWORDS • cancer • cytotoxic cells • immunogenic modulation • immunotherapy • low-dose radiotherapy • radiation • single-dose radiotherapy • T cells

The ability of ionizing radiation (IR) to influence the immune response against tumors has become more and more attractive as the development of novel cancer immunotherapies (CIT) has rap-idly expanded and these agents have come into wider clinical use. Beginning in 2010, Provenge (Sipuleucel-T), Yervoy (ipilimumab), Keytruda (pembrolizumab) and Opdivo (nivolumab) have all received US FDA approval for the treatment of cancer. While only four CITs have received FDA approval at this time, there are numerous others under preclinical study and in clinical development. As a result, the immune enhancing powers of radiation are becoming a valued aspect and important use of radiotherapy (RT) [1].

Radiation can be used as an adjuvant to immunotherapies in several ways. First, it can induce a type of cell death in a subset of susceptible tumor cells, which can then activate antigen uptake, cell maturation and presentation by antigen-presenting cells (APCs). This immunogenic cell death (ICD) is identified by three main hallmarks on tumor cells; calreticulin exposure, ATP release and HMGB1 release [2]. APCs responding to ICD can subsequently induce other immune cells that are capable of attacking the surviving tumor cells. This body of work has been highlighted in a number of excellent reviews [3–7]. Second, radiation can cause molecular alterations in tumor cells in a manner that directly sensitizes tumor cells to immune cell-mediated killing. This property of radiation is referred to as immunogenic modulation (IM) of tumor cells [8,9]. Pre-existing (endog-enous) immune cells, or those induced or activated by vaccine, may not be able to act once they reach tumors if the tumor microenvironment (TME) is immunosuppressive or the tumor cells themselves are suppressive or suboptimal targets. Modulated tumor cells surviving exposure to radiation, either because they are radio-resistant or because they receive sublethal doses, can become better targets

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for antitumor immune cells. Third, radiation can alter the activity and function of immune cells directly. Which of these three situations occurs is likely influenced by the dose and deliv-ery scheme used (single dose vs separated into smaller fractions), though they are likely not mutually exclusive.

The immune enhancing effects of RT, and the different ways that RT has been combined with CITs clinically and preclinically, have been recently reviewed elsewhere [10,11]. Exciting out-comes have been observed in patients receiving RT for palliation with no intent to cure [12,13]. Similar success has been reported when higher doses of RT were used [14–16]. These clinical outcomes are even more exciting because they report immune mediated regression of not only the irradiated tumor, but also of distant tumors outside of the radiation field (i.e., abscopal response). Though these abscopal responses and clinical outcomes are thrilling, there remains significant room for improvement [10]. It is not fully understood what induces such responses on the cellular and molecular level, and it is not yet possible to routinely recapitulate these outcomes.

The focus of this review is on IM of tumor cells and the influence of radiation dose on the phenotype of tumor cells. The use of radia-tion for IM would not rely on RT to induce a de novo immune response, but would instead be used to specifically complement the elabo-rate CITs already in development [17,18]. At this time, RT is not routinely incorporated into most CIT approaches specifically for its IM properties. Our review will consider the effect of both low doses of radiation (≤2 Gy; see the ‘Immunomodulation by low-dose radiotherapy’ section), and hypofractionated doses (2–25 Gy; see the ‘Immunomodulation by hypofraction-ated doses of radiation’ section), on gene expres-sion in cells surviving radiation, focusing on changes that can directly enhance cellular attack of tumor cells. We also consider the impact of these radiation doses directly on immune cells themselves both phenotypically (see the ‘Immunomodulation by low-dose radiotherapy’ and ‘Immunomodulation by hypofractionated doses of radiation’ sections) and functionally (see the ‘Immunomodulation at work’ section). Studies comparing responses to single dose (SD) versus multifraction (MF) delivery have been recently reviewed by others [19,20]. Here we leave our review to observations made in

tumor cells surviving SD radiation, or systems where r adiation alone has no observable tumor control.

●● Immunomodulation by low-dose radiotherapy – ≤2 GyIM of tumor cells following low-dose radiotherapy dosesThere are many reports on the ability of IR to modulate the expression of genes that are important for immune attack of tumor cells. However, much of the information available is from cells receiving high doses of radiation intended to kill tumor cells, and much less information is available from tumors treated with low or sublethal doses of radiation; yet, changes in several important immune genes have been reported in tumor cells treated with low-dose radiotherapy (LD-RT). MHC-I is responsible for presentation of endogenous antigens to cytotoxic T lymphocytes (CTLs). Reits et al. exposed a human melanoma cell line (MelJuSo) to radiation and quantified cell surface MHC-I complexes 18 h later. Radiation induced a dose-dependent increase in MHC-I expression in vitro and increases in MHC-I were observed beginning at 1 Gy. Furthermore, they observed more polyubiquitinated pro-teins available for proteasomal degradation after radiation doses as low as 1 Gy. This and other studies revealed that radiation can lead to more peptide diversity for MHC-I antigen presentation, in addition to enhanced MHC-I expression [21–23]. The stress ligands MICA/B can bind to cognate receptors (NKG2D) on cytokine-inducible lymphocytes, NK cells and T cells causing activation of these cells [24]. Liu et al. irradiated human myeloma cells (KAS-6/1) at various doses and analyzed the cells for expression of MICA/B 24 h postirra-diation [25]. A dose-dependent increase in both MICA/B expression at doses as low as 2 Gy was observed. After appropriate activation, CTLs and NK cells can kill tumor targets expressing death receptors such as Fas (CD95) on their surface, and there is some evidence of increased Fas expression following treatment of tumors with LD-RT. In one study, Sheard et al. treated human colorectal (HCT116) and human breast cancer (MCF7) cells with 2 Gy IR and observed moderate upregulation of Fas [26]. The ligand for Fas, FasL, sends apoptotic signals into Fas expressing cells. Abdulkarim et al. irradiated a human nasopharyngeal cancer (C15) cell line

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at various doses and measured FasL induc-tion [27]. Fas is constitutively expressed in this NPC cell line prior to irradiation, but follow-ing 2 Gy IR there was an increase in FasL on these cells. FasL expressing cells could possibly kill themselves and/or nearby Fas-expressing cells, including infiltrating T cells. This is a rare example of LD-RT upregulating expression of a gene that could negatively impact T cells.

Mechanism of IM in tumor cells following LD-RTRadiation has been paradoxically reported to be both proinflammatory and anti-inflammatory. As the key transcription factor for numerous immune factors, NF-κB has been investigated for its involvement in gene expression follow-ing LD-RT. The use of LD-RT to treat benign inf lammatory or hypoproliferative condi-tions is well known [28,29], and inhibition of NF-κB is thought to be responsible for these anti-inflammatory activities. In this regard, Lodermann et al. observed a decrease in p38 (an upstream molecule of NF-κB) and AKT (a downstream molecule of NF-κB) following 0.5 Gy and 0.7 Gy of IR. Pajonk and colleagues reported that the 26S proteasome was a direct target of IR, and hypothesized that radiation-induced inhibition of the proteasome was a mechanism of NF-κB inhibition. This type of inhibition could modulate expression of numer-ous proinflammatory molecules, such as TNF-α, IL-1β and IL-6, at the transcriptional level. Indeed, bladder cancer cells (ECV304) treated with 0.17–2 Gy induced a rapid, dose-depend-ent decrease in 26S proteasome activity. In turn, this inhibition prevented the phosphorylation, ubiquitination and subsequent degradation of the IκB regulatory complex leading to reduced translocation of NF-κB complex to the nucleus. While altered proteasome function induced by LD-RT is an attractive explanation for many of the observed transcriptionally regulated effects, via inhibition of NF-κB, more investigation is needed in this area as chronic low dose exposure has resulted in both stimulation and inhibition of NF-κB regulated genes [30]. Further, most studies have utilized doses higher than 2 Gy to investigate radiation-induced changes in gene expression in tumor cells and NF-κB activation is observed at higher doses. Thus IR appears to be able to both activate and inhibit NF-κB and the outcome may be dependent on dose, and perhaps tissue, under study.

Modulation of immune cells following LD-RTThe efficacy of RT is known to be influenced by the TME, including local expression of agents such as HIF-1 and VEGF [31]. However, the reciprocal is also true and LD-RT can directly modulate the efficacy of immune cells including macrophages, DCs, NK and lymphocytic cells. LD-RT in vitro (0.075 Gy) increases NK cell secretion of IFN-γ and TNF-α [32]. Nontumor bearing mice treated with 0.2 Gy × 4 total body irradiation (total 0.8 Gy) had an increase in NK and NK T cell numbers at 21 days post IR [33]. Increased amounts of IFN-γ, IL-12 and TNF-α from macrophages were detected 10 days post IR in these animals. Klug et al. observed that 2 Gy irradiation induced iNOS expression (and reduced HIF-1, Ym-1, Fizz-1 and arginase expression) indicating an induc-tion of the M1 (proinflammatory) macrophage phenotype [34]. They further showed that iNOS inhibition blocked VCAM-1 expression on CD31+ endothelial cells, indicating endothelial cell activation, and that the capacity to support leukocyte transmigration was enhanced post IR via iNOS. Lodermann et al. observed a decrease in IL-1β secretion from LD-RT-induced mac-rophages, and found that this downregulation was correlated with reduced nuclear transloca-tion of the p65 (RelA) subunit of the NF-κB complex [35]. In another study, direct treat-ment of macrophages with IR decreased IL-1β (0.5–2 Gy) and increased TGF-β (0.1–0.5 Gy) expression with a decreased in nuclear locali-zation of NF-κB also observed [36]. This dem-onstrates that LD-RT can change macrophage phenotype. Moreover, Liu et al. observed increased expression of CD80 and CD86 on macrophages following LD-RT suggesting an increased capacity for costimulation of T cells [37]. LD-RT can also impact other APCs such as DCs. Shigematsu et al. treated murine DCs with various doses of IR (0.02, 0.05, 0.1, 0.5, 1 Gy) and observed increased secretion of IL-12 and increased expression of IL-2 and IFN-γ mRNA at doses as low as 0.05 Gy from DCs [38]. They further observed that LD-RT had no effect on proliferation or maturation of the DCs. Whole body irradiation of non-tumor bearing mice with 0.075 Gy caused an increase in CD28 expression concomitant with a reduction in CTLA-4 expression on splenic lymphocytes [37]. Conversely, treatment with 2 Gy produced reciprocal changes in these same cells. Treatment of T

REGS from rats with 0.15 Gy

Future Oncol. (2016) 12(2)242



of radiation in vitro reduced the expression of CTLA-4 [39], which is associated with the sup-pressive function of these cells. These findings demonstrate that LD-RT can directly alter the state of some immune cells with important roles in antitumor immunity.

The ability of lower dose ionizing radiation (<0.2 Gy) to activate immune cells has been recently reviewed by Farooque et al. [40], and we now present a table summarizing important immune relevant changes in both tumor cells and immune cells following low to intermediate doses of IR in Table 1.

●● Immunomodulation by hypofractionated doses of radiationIM of tumor cells within the hypofractionated dose rangeDoses between 2 and 25 Gy have been evalu-ated extensively for enhancement of antitumor immune attack. In the context of radiation

biology, a radiation dose of 5 Gy is in the mod-erate hypo-fractionated range, and 10 Gy dose is considered in the extreme hypo-fractionated range [41]. Stereotactic body radiation therapy has been administered in fractions up to 15 Gy [42]. As a result, the majority of IM studies have been conducted within these dose ranges, and there is much information available about the diverse immune relevant genes that are changed post IR (Table 2). Though typically delivered in multiple fractions over time to reach a higher cumula-tive delivery dose to patients, here we review the impact of mostly single dose (SD) exposure on the modulation of surviving cells.

Signal 1CTLs must see target tumor-associated antigens (TAA) displayed in MHC-I, and several studies have shown that IR can modulate this signal within tumor cells. Lugade et al. demonstrated that irradiated tumors (15 Gy) had an increased

Table 1. Low-dose radiotherapy range (≤2 Gy).

IM gene Tissue (up/down) Dose (Gy) mRNA or protein

Role Ref.

Tumor cells

MHC-I Melanoma (Hu); Up 1 Protein Antigen presentation [21]

FAS CRC, BrCa (Hu); Up 2 Protein Transmits apoptotic signals into cells [26]

FasL NPC (Hu); Up 2 Protein Inducer of apoptosis [27]

NKG2DL (MICA/B) Myeloma (Hu); Up 2 mRNA/protein Increased recruitment and function of NK cells [25]

Immune cells

IFN-γ NK cells; Up 0.75 Protein Activation of CD8 T cells; Th1 promoting [32]

Macrophages; Up 0.8 mRNA [33]

DCs; Up 0.05 mRNA [38]

TNF-α NK; Up 0.75 Protein Inducer of tumor apoptosis [32]

Macrophages; Up 0.8 mRNA [33]

IL-12 Macrophage; Up 0.8–0.075 mRNA/protein Th1 promoting cytokine [33,37]

DCs; Up 0.05 mRNA/protein [38]

IL-10 Macrophages; Down 0.075 Protein Immunosuppressive cytokine [37]

IL-1β Macrophages; Down 0.5–0.7 Protein Proinflammatory action [35,36]

0.5–2 Protein TGF-β Macrophages; Up 0.1–0.5 Protein Inhibits CTL function [36]

iNOS Macrophage; Up 2 Protein M1 associated, inducer of cytotoxic mediator NO [34]

Arginase Macrophages; Down 2 Protein M2 associated enzyme [34]

HIF1 Macrophages; Down 2 Protein Proangiogenic; tumor promoting [34]

CD80/86 Macrophages; Up 0.075–2 Protein Increased T-cell costimulation [37]

IL-2 DCs; Up 0.05 mRNA T-cell proliferation cytokine [38]

CD28 Lymphocytes; Up Lymphcytes; Down

0.075 2

Protein Protein

Transmits positive signal into T cells [37]

CTLA-4 Lymphocytes; Down Lymphocytes; Up

0.075 2

Protein Protein

Inhibitory signal for cytotoxic cells [37]

TREGS; Down 0.15 Gy Protein [39]BrCa: Breast carcinoma; CRC: Colorectal carcinoma; CTL: Cytotoxic T lymphocyte; DC: Dendritic cell; Hu: Human; Ms: Mouse; NPC: Nasopharyngeal carcinoma.

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Table 2. Hypofractionated dose range (>2 to <25 Gy).


Role Ref.

Signal 1

MHC I Mel (Hu, Ms); Up Mel (Ms); Up CRC, Lu, PCa (Hu); Up Glioma (Ms); Up PCa (Hu); Up

1–25 15 10–20 4 25

Protein Protein Protein Protein Protein

Antigen presentation

[21] [43] [44] [45]

[46]

TAA peptides (Hu, Ms); Up 10–25 Peptide saturation

T-cell target [21]

TAA Tumor lines (Hu); Up 10–20 Protein [44,46–47]

Effector signals

OX40L, 4-1 BBL CRC lines (Hu); Up PCa Lines (Hu); Up

10 5–15

mRNA/ protein Protein

Positive co-stimulation [48] [49]

CD70, ICOSL PCa lines (Hu); Up 5–15 Protein Positive co-stimulation [49]

PD-L1 PCa; Down BrCa tumor (Ms-TUBO); Up PCa tumor; Up/down

5–15 12 10

Protein Protein mRNA

Inhibition of T cells

[49] [50] [51]

CTLA-4 PCa tumor (Hu); Down 5–15 Protein Inhibition of T cells [49]

ICAM-1 CRC, Lu, PCa, HN (Hu); Up BrCa (Ms); Up BrCa (Hu); UpTumor lines (Hu); Up

10–202 × 12 Gy 10 10–20

mRNA protein Protein Protein mRNA/protein

Mediates leukocyte adhesion to tumor cells

[44,52] [53] [47] [54]

MICB, ULBP1/2 Tumor lines (Hu); Up 20 RNA Protein

NKG2DLs, trigger cytotoxic cells [55]

RAE-1 BrCa tumor (Ms); Up 2 × 12 Gy Protein NKG2DL, trigger cytotoxic cells [53]

Death receptors

FAS

Tumor lines (Hu); Up Tumor line (Ms); Up BrCa, CRC (Hu); Up Esophageal lines; Up

10–20 8–10 10–16 3–6

Protein Protein Protein Protein

Transmits death signal into cells

[44] [56]

[26,57] [58]

DR4 CRC (Hu); Up CRC, Lu (Hu); Up

2.5–10 10

Protein Protein

[59] [60]

DR5 CRC (Hu); Up 2.5–10 Protein [59]

Cytokines

TGF-β1 PCa tumor (Ms); Up 6–10 mRNA/protein Immunosuppressive [61]

GMC-SF, IL-6 PCa tumor (Hu); Up Lu (Hu); Up

10 25

Protein Protein

DC maturation [51,62]

TNF-α Sarcoma (Hu); Up 5 Protein Inducer of apoptosis [63]

Chemokines

CXCL16 BrCa (Ms); Up 2 × 12 Gy Protein Recruits immune cells [64]

CCR2, CCL2, CXCR6, CXCL16 HPV tumor; Up 14 mRNA [65]

Immune cells

CD70 DCs (Hu); Up DCs (Ms); Up

10 10

Protein Protein

Costimulates T cells [66] [67]

CD86

DCs (Hu); No change DCs (Ms); Up Macrophages (Hu); Up

10 10 2–20

Protein Protein Protein

Costimulates T cells [66] [67] [68]

CD40 Macrophages (Hu); Up 2–20 Protein Costimulates T cells [68]†Dose outside the 2–25 dose range of the other studies.BrCa: Breast carcinoma; CRC: Colorectal carcinoma; DC: Dendritic cell; HN: Head and neck; HPV: Human papilloma virus; Hu: Human; iNOS: Inducible NO synthase; Lu: Lung; Mel: Melanoma; Ms: Mouse; NO: Nitric oxide; PCa: Prostate cancer.

Future Oncol. (2016) 12(2)244



capacity for presenting antigen to specific T cells in an MHC-dependent manner [43]. RT upregu-lated MHC class I expression to high levels on glioma cells in mice [45]. Reits et al. observed a dose-dependent increase in MHC class I sur-face expression 18 h post IR in vitro in a human melanoma cell line (MelJuSo) at doses as high at 25 Gy [21]. Further, local irradiation (25 Gy) of mice in vivo resulted in similar increases in MHC within the irradiated tissue 24 h post IR. Additionally, cells exposed to 4 Gy IR exhibit increased TAP mobility when analyzed 1 h later and increased polyubiquitination follow-ing exposure to 10 Gy, indicating increased pro-tein degradation. Further analysis revealed that prolonged peptide saturation levels are directly related to increased radiation dose [21]. In vitro, human colorectal, lung and prostate cancer cell lines showed increase expression of various surface molecules, including MHC-I [44], 72 h post irradiation (10 and 20 Gy). Eight of the 23 lines increased surface expression of MHC-I and 17 of 23 increased surface expression of one or more of the TAAs evaluated (CEA or MUC). Gameiro et al. examined radiation’s ability to induce IM of human breast cancer (MDA-231), non-small-cell lung cancer (H522) and prostate cancer cells (LNCaP) 72 h after 10 Gy IR. Radiation significantly induced the surface expression MHC class I and TAAs in these diverse cell lines [47].

Signal 2Following recognition of antigen in MHC-I, naive T cells must also receive co-stimulatory signals for full activation and expansion. Once

activated, the activity and function of effector T cells are also enhanced by signaling through co-stimulatory molecules such as CD27, ICOS, OX-40 and 4–1BB [71]. Conversely, the actions of effector T cells can be inhibited by co-inhibi-tory signals through CTLA-4 or PD-1. We have recently demonstrated that colorectal tumor cells surviving 10 Gy radiation have increased mRNA and surface protein expression of both OX-40L and 4–1BBL [48]. This observation was further extended to prostate cancer cells, and an increase in the surface expression of 4–1BBL, ICOS-L, OX-40L and CD70 was observed in three human prostate cancer cell lines post IR [49]. Changes in OX-40L and 4–1BBL were dose-dependent (5–15 Gy) and increased expression was observ-able even after exposure to 15 Gy. By contrast, a decrease in the expression of the inhibitory molecule PD-L1 after exposure to 10 Gy IR was observed in some of these same cells, again in a dose dependent manner. Interestingly, reduced expression of PD-L1 remained stable even when evaluated 6 days post irradiation. Variable modulation of CTLA-4 expression in tumor cells (normally expressed on T cells them-selves) was reported. One of the tumor cell lines decreased CTLA-4 expression (DU145), while the other cell lines increased expression (PC3 and LNCaP). In contrast to tumor cells, normal prostate cells (PrEC) exhibited much less modu-lation of the genes evaluated. Aryankalayil et al., assessed the immunomodulatory changes in three human prostate cancer cell lines following exposure to 10 Gy fractionated (1 Gy × 10) or single dose (10 Gy × 1) radiation [19]. They found the mRNA for a variety of immune-related genes

Table 2. Hypofractionated dose range (>2 to <25 Gy).


Role Ref.

MHC-II Macrophages (Hu); Up 2–20 Protein Antigen presentation [68]

IL-12 DCs (Hu); Up 10 Protein Promotes Th1 responses [66]

IL-23 DCs (Hu); Up 20 Protein Promotes Th1 responses [66]

FoxP3 TREGS (Hu); Down 7.5–30 Protein TREG transcription factor [69]

CD45RO TREGS (Hu); Down 7.5–30 Protein TREG activation marker [69]

CD62L TREGS (Hu); Down 7.5–30 Protein Promotes trafficking to lymph nodes [69]

TGF-β1 TREGS (Hu); Down 30† Protein Immunosuppressive [69]

PD-L1 DCs, macrophages; Up 12 Protein Inhibitor of T cells [50]

Arginase and COX-2 Macrophages; Up 25 RNA/protein M2 associated, cancer promoting [70]

iNOS Macrophages; Up 25 RNA/protein M1 associated, inducer of cytotoxic mediator NO

[70]

†Dose outside the 2–25 dose range of the other studies.BrCa: Breast carcinoma; CRC: Colorectal carcinoma; DC: Dendritic cell; HN: Head and neck; HPV: Human papilloma virus; Hu: Human; iNOS: Inducible NO synthase; Lu: Lung; Mel: Melanoma; Ms: Mouse; NO: Nitric oxide; PCa: Prostate cancer.

245



to be modulated 24h post IR. Among the mod-ulated genes only PD-L1 had an obvious and direct link to cell-mediated attack against tumor cells. Exposure to 10Gy SD radiation induced the downregulation of PD-L1 gene expression in PC3 cells. It is important to note that multi-fraction (MF) delivery (1 Gy × 10) had the oppo-site effect in DU145 and caused upregulation of PD-L1 mRNA. No change in expression of this gene was reported in LNCaP. These data are in contrast to Bernstein et al., using SD, showing down-modulation of the protein from the cell surface at a later time post IR. Collectively, these studies could be highlighting major differences in IM response between SD and MF delivery. In the clinic, radiation is delivered in multiple smaller fractions to spare normal tissue, and the range of IM likely depends on fractions and frac-tion doses given [51]. In vivo, mice bearing breast cancer tumors (TUBO) were locally irradiated with 12 Gy of RT and PD-L1 expression was increased in tumor cells [50]. Overall, expres-sion of these co-stimulatory and co-inhibitory molecules is particularly interesting because it suggests that modulated tumors are not simply rendered more sensitive to attack by T cells but may be signaling back into responding immune cells and modifying their biological response.

Cell adhesion molecules such as ICAM-1 can enhance CTL and NK cell interaction with target cells, and also appear to deliver co-stimulatory signals into the responding cells. More than half of 23 diverse human tumor cell lines evaluated (colorectal, lung, and prostate) increased expres-sion of ICAM-1 following 10–20 Gy of external beam radiation (EBRT) [44]. Similar observa-tions have been seen in other cancer cell types including breast [47], head and neck [52], and skin [54]. Apart from EBRT, Chakraborty et al. have reported that exposure to a radionucleo-tide can also alter the phenotype of tumor cells. Samarium-153-EDTMP, a bone-seeking radio nucleotide, is used in palliation of bone metas-tasis. This study demonstrated that exposure to 153Sm-EDTMP induced several immunostimu-latory molecules, including ICAM-1, on pros-tate and lung cancer cells. Importantly, IM of ICAM-1 has also been reported 48 h after in vivo local RT (12 Gy × 2) in mice bearing breast tumors (4T1) [53].

Radiation has also been shown to modulate the expression of stress ligands that stimulate NK cell activation. These ligands (MICA/B and ULBP in humans and RAE-1 in mice) bind

to NKG2D receptors on NK cells (and CD8+ T cells) and stimulate their lytic activity [72]. Kim et al. observed an increase in MICB and ULBP1/2 following 20 Gy radiation in various cancer cell lines (melanoma, colon, cervical and lung) [55]. Increased expression of RAE-1 has similarly been reported following in vivo RT of tumor bearing mice [53].

In addition to antigen presentation and acti-vating signals to cytotoxic immune cells, the expression of mediators of effector activities such as death receptors, can also be modu-lated in tumor cells by IR. Chakraborty et al. observed a marked increase in surface Fas expression in MC38-CEA+ murine tumors treated with 8 Gy [56]. There are many reports of increased expression of Fas in human tumor cells as well [26,44,57–59]. Increased sensitivity to the receptors for TRAIL-mediated apoptosis has been reported post IR [73,74], and it has fur-ther been demonstrated that expression of both DR4 and DR5 can be modulated by radiation (2–10 Gy) in human tumor cells [59,60].

Cytokines can enhance immune activities of immune cells responding to tumor cells, and chemokines can facilitate the recruitment of immune cells into tumor tissue. IR can also modulate the expression of these molecules in tumor cells. Cytokines such as GM-CSF and IL-6 have been reported to be increased in the supernatants of two of three prostate cancer cell lines 48–72 h post IR (10 Gy) [51]. Another study reported an increase in the levels of GM-CSF, IL-1α and IL-6 in a human lung cancer cells line (AOI) 24 h post IR [62]. TNF-α was increased after treatment with 5 Gy in 5 of 13 human sar-coma cells [63]. Most recently, Wu and colleagues witnessed a significant increase in TGF-β1 in murine prostate cancer cells (TRAMP) both in vitro (6 Gy) and in vivo (10 Gy), suggesting that this immunosuppressive cytokine may also be induced from some tumors [61]. In addition to cytokines, IR can also alter the expression of chemokines from some tumor cells. The upreg-ulation of CXCL16 (CXCR6 ligand) within tumors and blood vessels of 4T1 tumor-bearing mice following local irradiation (12 Gy × 2) in vivo has been demonstrated. CXCL16 is quickly shed by 4T1 tumor cells and markedly increased the recruitment of CXCR6+CD8+ T cells into the tumor [64]. A significant increase in CCR2 and CCL2 mRNA was observed in HPV-associated tumors of mice locally irradi-ated with 14Gy [65]. CCR2 mediates monocyte

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chemotaxis and CCL2 recruits monocytes, MDSCs and DCs to sites of tissue injury and inflammation. The authors also reported signifi-cant upregulation of CXCR6 (regulates metasta-sis and progression of cancer via interaction with soluble CXCL16) and CXCL16 (induces migra-tion of several subsets of T cells and NK cells) after radiation. Collectively, these results reveal that hypo-fractionated doses of IR can modu-late soluble signals that influence the behavior of diverse immune cells as well as chemokines and ligands involved in homing of immune cells. Overall, the reports of phenotypic changes induced in tumor cells receiving the doses of radiation discussed in this review are summa-rized in Figure 1, and are overwhelmingly posi-tive in favor of promoting effective an titumor immune responses.

Mechanism of modulation in tumor cells within the hypofractionated dose rangeNF-κBSeveral mechanisms have been described for how IR can modulate expression of immune relevant genes in tumor cells (Figure 2). While LD-RT has been shown to reduce NF-κB via protea-some inhibition, hypofractionated doses of IR have conversely demonstrated increased activity of NF-κB. NF-κB can be activated in differ-ent cell types within an irradiated tumor mass including the tumor cells, stromal cells, cells of the vasculature, and immune cells. In tumor cells, genotoxic stress induced NF-kB activation is initiated by DNA double strand breaks (DSB) in the nucleus and can stimulate gene expression. NF-κB activation has been reported to occur in this manner in diverse tumor cell lines in an ATM-dependent pathway, mostly at doses above 3 Gy. ATM signaling alone is not sufficient; post-translational modification of NEMO is needed, and is also induced by IR. Fully composed ATM-NEMO complexes (reviewed in [30]) can phos-phorylate IkB resulting in its degradation by the 26S proteasome. This frees NF-κB to translo-cate into the nucleus and activate NF-κB target genes. Subsequent expression of NF-κB-induced cytokines can sustain this signaling pathway in a positive feed forward loop. Not surprisingly, some of the genes modulated by IR in tumor cells, as mentioned above, are known NF-κB target genes (MHC-I, ICAM1, cytokines) [30]. ATM signaling is also a well-known activator of p53. p53 target genes, however, are related mostly to cell cycle, apoptosis and DNA repair

pathways (reviewed in [75]). While most p53 inducible genes are not typical immune relevant genes, apoptosis inducing death receptors could be important for immune cell mediated signals transmitted from death ligands on cytotoxic immune cell. Unfortunately, less than 50% of tumor cells retain functional p53.

mTORWhile NF-κB may be responsible for MHC-I upregulation early post IR, the kinase mTOR has been shown to be responsible for the radi-ation-induced increase in MHC-I expression at later times post IR (24 h) [21]. mTOR is a critical regulator of protein translation by caus-ing enhanced ribosomal translation. As overall translation is increased, so is the intracellular pool of peptides available for binding to MHC-I. This increase in peptides appears to be respon-sible for the increased MHC. Interestingly, the benefit of combination immunotherapy with RT was lost when an mTOR inhibitor was utilized demonstrating the therapeutic relevance of this pathway post IR [74].

EpigeneticIt has recently been reported that IR-induced DNA hypomethylation induced gene expres-sion in CRC cells treated with 2–5 Gy [76]. Our studies have found that increased 4–1BBL expression following 10 Gy radiation is medi-ated by increased histone acetylation at the pro-moter for this gene in human CRC cells [48]. More recently, we have reported that radiation (5 Gy) similarly increases histone acetylation at both the Fas and DR5 promoters in addi-tion to the 4–1BBL promoter. Further, less HDAC2, HDAC3 and DNMT1 were bound to the promoter regions of both 4–1BBL and Fas, but not other genes following IR of human tumor cells [77]. These observations suggest that radiation can also regulate the expression of specific genes by epigenetic mechanisms. This could be important for combination radiation- immunotherapy approaches, as epigenetic changes can be maintained for quite some time in the cell population. At this time, it is unclear how IR is altering the binding of these enzymes at specific gene promoters.

miRNAsmiRNAs are key regulators of gene expression. Several miRNAs have been reported to be modu-lated following exposure to IR [78,79]. There is

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also evidence for regulation of gene expression via miRNAs in normal endothelial cells exposed to IR [80]. Interestingly, miR16 was shown to be downregulated following exposure to IR (10 Gy delivered in fractions), and 4-1BBL (TNFSF9) was identified as one of the target genes whose expression was increased in response to this miR-NAs reduction. If this same mechanism of regu-lation operates in irradiated tumor cells needs

to be determined. These data highlight the fact that molecules co-stimulatory to effector T cells seem to be induced by radiation in a variety of cell types, including both normal and tumor.

Modulation of immune cells within the hypofractionated dose rangeCells of the immune system can be rapidly dividing, and therefore vulnerable to radiation.

Figure 1. Immunogenic modulation of tumor cells by ionizing radiation. Tumors have been reported to be modulated in several ways, which could directly enhance the function, activity or recruitment of CD8+ T cells, as well as the function of NK and dendritic cells. Increased TAA could result in increased presentation on MHC-I to effector cytotoxic T lymphocytes but could also make more antigen available for uptake by antigen-presenting cells. There have also been limited reports of modulation of tumors in a manner that could negatively impact CD8+ T-cell activities. Note: Ionizing radiation-induced immunogenic cell death mechanisms that enhance dendritic cell activities are not depicted in this figure. †PD-L1 has been reported to be both increased, and decreased, by ionizing radiation. Ag: Antigen.

?

Ionizing radiationDendriticcell

Positive immunogenicmodulation todendritic cells GM-CSF

++

+

CD8+

TCR

+

CD8 T cells– Endrogenous /pre-existing– Induced/activated via vaccine– Adoptively transferred in

CD8+

Positive immunogenic modulation to CD8+ T cells

CD8+

Negative immunogenicmodulation to CD8+ T cells

TregsCD8 cell function

TGF-βPositive immunigenicmodulation to NK cells

NKG2D

NK cell

MICA/BULBP1/RAE-1

PDL1†

FasL

Cytolytic activityCALRDR4

DR5Fas

MICA/BULBP/RAE-1

TAA

CD704-1BBLOX-40L

ICAM-1MHC 1/agICOSL

SurvivalActivation

Signal 1

Tumor cell

More Agavailable forphagocytosis

CXCL16

TNF-α

CCR2, CCL2,CXCR6

IL-1α

IL-6

Immunogenic modulationof soluble factors and endothelial cells

Vasculature

VCAM

CXCR6+

CD8s

Future Oncol. (2016) 12(2)248

Figure 2. Diverse mechanisms of immunogenic modulation reported in tumor cells surviving radiation. †NF-κB has been reported to be both inhibited and activated at lower radiation doses depending upon tissue and length of exposure.

<2 Gylow radiation doses

26S proteosome

NF-κB activation

Reduced expressionof NF-κB target genes†

>2 to <25 Gyhypofracted radiation doses

mTOR

Enhancedribosomal translation

DNAdouble-strand break

ATM

MEMO

NF-κBactivation p53

H3

AC

HDAC2/3 DNMT1at some gene promoter sites

MHC-1 NF-κB targetgenes:– MHC-I– ICAM-1– Cytokines

p53 induciblegenes:– Apoptotic e.g., Fas– DNA repair– Cell cycle

4-1 BBLFas



Individuals receiving heavy doses of radiation (atomic bomb survivors) exhibit severe damage to mature lymphocytes and bone marrow stem cells, resulting in depletion of several immune cell sub-sets [81]. This has led to the long-standing percep-tion that radiation beyond the low dose range is generally immune suppressive. In recent years, however, there have been several reports of higher dose radiation treatment directly modulating the phenotype of immune cells both positively and negatively.

Evidence demonstrating positive modula-tion of immune cells, in a manner that would make them more effective for antitumor activ-ity, has been accumulating and is summarized in Table 2. For example, Huang et al. observed the upregulation of CD70 on mature den-dritic cells and CD20+ B cells isolated for human PBMCs following exposure to 10 Gy in vitro [66]. Further analysis showed no change in CD80 and CD86 expression or the maturation marker CD83 on DCs. Irradiation of DCs also increased IL-12 and IL-23 cytokine secretion with doses as high as 10 Gy and 20 Gy, respec-tively. Following local irradiation (10 Gy) of

mice bearing B16 tumors, Gupta et al. detected significant upregulation of CD70 and CD86 on live DCs (CD45+CD11chighMHC-IIhigh) by flow cytometry 48 h post RT [67]. Radiation has also been reported to increase markers of activation in monocytes [68]. In vitro treatment with 20 Gy (and 2 Gy to a lesser degree) induced significantly higher expression of CD40, MHC-II (HLA-DR) and CD86 48 h post IR on the monocyte cell line U937 in an NF-κB dependent manner. APCs modulated in such ways would be better able to stimulate subsequent antitumor immune responses.

Conflicting data exist regarding the radiosensi-tivity of different subsets of lymphocytes. Thus, it is very important to elucidate how different com-ponents of the immune system are altered by IR. In particular, there are many conflicting reports on the impact of radiation on CD4+ T

REGS, which

play a major role in immune tolerance [82]. In cancer patients, T

REGS inhibit the generation of

successful antitumor immune responses and increased infiltration of T

REGS is often detected

within the TME [83,84]. One study suggests that irradiation may modulate the phenotype

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(and function) of human TREGS

in vitro [69]. CD4+CD25+ T

REGS from PBMCs were irradi-

ated and evaluated for expression of TREG

spe-cific markers. There was reduced expression of CD62L, FOXP3 and CD45RO and increased expression of GITR 12 h post irradiation (1.875, 7.5 and 30 Gy). By contrast, there was no change in the expression of CD4, CD28 and CD45RA on T

REGS. The expression of membrane TGF-β,

a suppressive effector molecule of TREGS

, was also decreased after irradiation of T

REGS with 30 Gy

as compared with nonirradiated TREGS

. Here, reduced expression of T

REG effector molecules

could correlate with reduced suppressive activ-ity thus allowing effective antitumor immune responses.

There have also been reports of radiation modulating immune cells in a manner that could be inhibitory to effective antitumor immune responses. After exposing TUBO breast cancer cells to 12 Gy RT, PD-L1 expression was increased in DCs after 72 h [85]. There was also a slight increase in PD-L1 seen on macrophages but no observable change in MDSC expression of PD-L1. Curiously, PD-1 expression on CD8+ T cells was slightly reduced 3 days post-irradia-tion in this same study. It is unclear, however, if this was direct modulation by RT or modulation in response to factors produced by other cells in the local environment. Irradiation of mice bearing murine prostate tumors (TRAMP-C1) induced phenotypic changes in macrophages associated with cancer promotion [70]. Higher dose RT with 25 Gy induced increased arginase and COX-2 and promoted tumor growth [70]. iNOS was produced at later times in these cells and at low levels. This is the opposite of findings discussed above, by Klug et al. using LD-RT, and highlights the importance of understanding the impact of IR dose on immune cell activities. The impact of radiation specifically on MDSC phenotype has not been extensively studied, but there are reports of both expansion [86] and reduction [87] in MDSC numbers post IR.

●● Immunomodulation at work (functional outcomes)In vitro evidence of IM & functional enhancement of cytotoxic immune cellsIt is clear that there is much more to RT than its traditional use for direct tumor cell destruc-tion or palliation of pain. IR has the ability to modulate gene expression in tumor cells in ways that make them better stimulators of immune

cell function [9]. This ability of IR has been dem-onstrated in several in vitro model systems where the function of immune cells is enhanced as a direct consequence of interactions with irradi-ated tumor cells. Sublethal irradiation of colo-rectal carcinoma cells (CRC) lines resulted in enhanced susceptibility to lysis by tumor specific CTLs following 10 Gy of irradiation [44]. CTL killing was MHC restricted as irradiated MHC mismatched CRC were not killed by the TAA specific T cells. Moreover, increased killing by CTLs was also observable against other cancer types including prostate [47] and head neck squa-mous carcinoma [52], revealing that enhanced killing of irradiated tumor cells is not restricted to a single cancer type. More recently we have reported that interaction of CTLs with irradi-ated human CRC cells enhances the survival and activation of T cells [48]. Enhanced CTL func-tion against irradiated tumor cells is not limited to EBRT, as Chakraborty et al. have reported that exposure to 153Sm-EDTMP enhanced CTL killing of irradiated prostate tumor cells (LNCaP) [46]. Despite different molecular pro-files of IM, studies by Gameiro and colleagues reveal that radiation-induced tumor sensitivity to CTL lysis was equally augmented with single or fractionated doses of radiation, suggesting that either regimen could elicit effective attack by T cells [47]. In addition to modulated tumor cells, immune cells modulated by RT also have the ability to enhance T cell function. For example, irradiated DCs have been shown to increase the proliferation and IFN-γ production from T cells as a direct consequence of increased expression of CD70 [66]. There is also some evidence that the ability of T

REGS to suppress cells is reduced

following both high-dose [69] and low-dose i rradiation in vitro [39].

IM of tumor cells by RT has also been shown to enhance functional activity of NK cells. NK cell-mediated recognition and cytolysis of human tumor cells is governed by ligation of the NKG2D receptor on NK cells with MICA and MICB (MICA/B) molecules on tumor cells. Human melanoma cells and non-small-cell lung cancer cell lines that increase expression of MICA/MICB after 20 Gy IR also exhibited enhanced sensitivity to NK mediated killing [55]. Liu et al. reported that upregulation of MICA/B on the tumor cell surface led to increased recruitment, activation and sur-vival of NK cells [25]. Direct exposure of human NK cells to radiation can result in increased NK cytotoxicity that seems to peak at an optimal

Future Oncol. (2016) 12(2)250



dose (6 Gy) and decline after the peak (range: 1–16 Gy). Radiation-treated NK cells from can-cer patients have been reported to have higher lytic activity than NK cells from normal controls [88]. Radiation also enhanced the cytotoxic effects of NK cells when they were inoculated, as a mixture with tumor cells, into mice after irradiation [89]. This synergistic effect was not observed when the lymphoid cells were inoculated after irradiation, indicating that lymphoid cells needed to be in con-tact with tumor cells before irradiation. Anti-asialo GM1 reversed this effect implicating NK cells as the effectors here. Purified NK cells exposed to 0.2 Gy LD-RT in vitro displayed no significant dif-ference in cell viability or proliferation. However, significant augmentation of cytotoxic function was detected when NK cells were stimulated with low-dose IL-2 prior to irradiation [90]. Others have similarly reported that radiation-treated NK cells exhibit increased function [91–93]. Collectively, these studies demonstrate the ability of IR to enhance NK cell activity by both direct treatment with IR and as a consequence of radiation-induced IM of the tumor.

In vivo evidence of IM & functional enhancement of cytotoxic immune cellsAll cells in the TME, including the target tumor cells, endothelial cells, stromal cells and infil-trating immune cells, may respond uniquely and further influence the response of neighbor-ing cells. While immune cells are thought to be more radiosensitive than other cells in the TME, there are numerous accounts of local tumor irra-diation in vivo culminating in effective immune attack of tumors by both adaptive and innate cells. RT has been shown to enhance tumor-specific CTL numbers and function in the intact TME. Chakraborty et al. demonstrated enhanced killing of CEA+MC38 tumors by CEA specific CD8+ T cells in mice irradiated with 8 Gy. The abil-ity of RT to enhance attack of tumor cells was demonstrated using both a therapeutic vaccine to induce T cell expansion [94] as well as when tumor specific T cells were adoptively transferred into the animals [56]. In the latter study, modula-tion of the Fas receptor on tumor cells was shown to be required for the ability of RT to enhance immune attack. Using another tumor model system, adoptive cell transfer of ex vivo activated OVA-specific OT-1 CD8+ T cells led to increased infiltration of transferred T cells to tumor sites (and not merely expansion of localized T cells) following RT (15 Gy) to the tumor [95]. This

study suggested that the mechanism of radiation-induced lymphocyte infiltration into the TME involved upregulation of chemo-attractants MIG and IP-10. Though it was not determined which cells were producing these factors, these chemo-attractants appeared to promote IFN-γ responses by conditioning the tumor microenvironment for enhanced CTL trafficking. Moreover, the tumor-infiltrating lymphocytes (TILs) isolated from in vivo irradiated tumors killed better than TILs from no nirradiated hosts.

There are similar reports of radiation modulat-ing tumor cells in vivo and enhancing tumor-spe-cific NK cell numbers and function. Ruocco et al. demonstrated that RT (12 Gy × 2) synergized with CTLA-4 blockade by upregulating RAE-1 expression on the surface of irradiated tumor cells and that the therapeutic effect of the combinato-rial regimen were blocked by antibodies targeting the RAE-1 receptor (NKG2D) [53]. IL-12/1L-15/1L-18 preactivated NK cells showed increased frequencies and persistent effector functions inside established murine tumors following RT (5 Gy TBI), highlighting the enhanced therapeu-tic efficacy of combination NK cell therapy and RT [96]. In patients, NK cells from uterine cer-vix carcinoma patients undergoing radiotherapy demonstrated increased cytotoxicity, suggesting that it is possible for RT to enhance immune cell function in the human TME [97].

RT can also influence other noncytotoxic immune cells in a way that could positively influ-ence antitumor attack. There have been limited reports of RT, in the dose ranges discussed here, modulating APCs in the TME to enhance cross-presentation of TAA [98] or recruiting additional immune cells including CD8+ T cells into the TME [99]. T

REG mediated suppressive activity

from irradiated (8.5 Gy) melanoma (D5) tumor bearing mice has been reported to be signifi-cantly reduced when compared with T

REGS from

untreated mice indicating that RT impairs func-tion of T

REGS [100]. However, there have also been

contrasting reports revealing that RT (10 Gy) can induce suppressive immune cells following local RT to some tumors (TRAMP) [61]. Thus, it remains unresolved if IM of tumors by radiation can directly influence T

REG number and function.

ConclusionThe most exciting reports of RT-CIT in com-bination have been of the abscopal response following RT both preclinically [101,102] and clinically [12–16,103]. The abscopal response likely

251



involves cytolytic cells that retain the functional ability to mount a sustained response at a distant, nonmodulated, tumor site. The mechanism(s) of the abscopal effect has not been definitively determined. Molecules that can alter the lytic capacity or enhance the sustainability of effec-tor CTLs or NK cells are likely candidates for promoting this type of effect. For a long time increased expression of death receptors such as Fas was thought to be the sole molecular mecha-nism responsible for enhanced immune attack of irradiated tumors [56]. However, very simple in vitro systems demonstrated that human tumor cells with deficient Fas signaling were still killed better by CTLs after radiation suggested that alternate mechanisms exist and contribute to this effect [44]. Use of these in vitro systems has allowed us to directly test the impact of irradiated tumors on CTL biology, and has revealed that CTLs also survive better and are activated better follow-ing interaction with irradiated tumor cells [48]. Examination of irradiated tumor cells for changes in expression of genes that could provide survival, activation and enhanced lytic activity in effector CTLs revealed that the co-stimulatory molecules OX-40L and 4–1BBL were upregulated on irra-diated tumor cells [48,49]. Increased MHC-I and antigen expression following IR have been dem-onstrated by several groups. Increased signal 1, however, must work in concert with other signals to T cells. Increased antigen in a toleragenic or immunosuppressive environment where robust costimulation is not present leads to suboptimal immune responses such as T-cell anergy. Thus, many proposed mechanisms of RT-enhanced immune responses (more antigen presentation, immunogenic death, increased death receptor, increased stress ligands or increased cell adhe-sion expression) would still require appropriate co-stimulation to induce optimal and sustain-able CTL responses. Furthermore, these changes all occur local to the irradiated tumor and it is difficult to envision how many of the reported changes at the local irradiated tumors could pro-mote a response that is sustainable at a distant tumor unless the change was retained within the responding effector cells. Receiving signals from OX-40L or 4–1BBL, or perhaps effector activity programming cytokines, from irradi-ated tumor cells could leave a lasting impression on T cells leaving the tumor and impact future activity beyond the irradiated tumor [104,105]. This argument seems plausible given the fact that the mere presence of antigen specific T cells (or

induction of greater numbers by vaccine) is not enough to induce curative T cell activity against many tumors. Indeed, there are situations where T cells of known TAA-specificity already exist but are not effectively attacking the tumor, even when large numbers are transferred into tumor bearing hosts [56]. In these situations radiation is not needed to ‘prime’ an initial T cell response to the TAA via ICD because large numbers of effector cells are being transferred in. Local RT does, however, result in greatly enhanced activity of T cells suggesting that the existing T cells may now be receiving different signals from the irradi-ated tumor [43,106]. Moreover, irradiated tumor cells alone, and in the absence of APCs, are killed better by effector CTLs [44,46–47,52], and are able to enhance T cell survival and activation [48]. This mechanism would be relevant when effec-tor CTLs are already present but have less than optimal functional or killing capacity.

At this time, RT is routinely applied for pal-liation or tumor debulking in patients where it is not curative. Given the substantial evidence of the IM activities of IR, perhaps now is the time to begin utilizing RT specifically for IM in combo with CIT strategies. Though this review focused on radiation doses of 25 Gy or below, the influence of various doses within this range should be considered a critical factor moving forward [107,108] in defining the most effective dose for promoting IM activities. For example, NK cells are sensitive to high doses of radiation while lower dose radiation enhances cytotoxicity of NK cells [93]. Lower doses may help achieve a more favorable balance between tumor-infiltrat-ing cytotoxic cells, and the unfavorable suppres-sive cells, allowing for tumor elimination [109]. Many RT-CIT studies use much higher doses (>25 Gy total) [87,110–111], and it is unclear if the immune outcomes reported occur similarly at lower doses. To this point, many differences are seen in the modulation of MHC-I following low dose [45] versus higher dose [112]. Lower doses may also be fundamentally better for use as an adjuvant to CIT because of lower toxicity as well as reduced clinical visits for the patient. In addi-tion to dose range, single dose delivery versus multiple fraction delivery seem to differentially influence IM outcomes [19,20].

Future perspectiveWhat is next regarding immunomodulation and radiation therapy for cancer? IM-induced phenotypic changes in tumor cells have been

Future Oncol. (2016) 12(2)252



studied extensively. However, given the differ-ential radio-sensitivities of immune cell subsets, it seems imperative to more fully elucidate the direct effects of LD-RT and radiation doses within the hypofractionated dose range on immune cell function. It will also be important to determine which phenotypic changes occur most broadly and can thus be capitalized on across diverse human tumor types. Regarding the TME, how stromal cells and local endothe-lial cells are modulated and contribute to anti-tumor activity or immune suppression at these doses needs to be elucidated both in vitro and in vivo. Given the recent evidence that IM of some genes is occurring via epigenetic mecha-nisms, determining how long these changes are retained in tumor cells as well as what other immune relevant genes are regulated this way will be helpful for defining the therapeutic use-fulness of RT-induced IM. It is perhaps most important to determine which changes occur in instances where abscopal responses are seen. The mechanisms reported for induction of the absco-pal effect are diverse, coming from both tumor immunologists and radiation biologists, and it will be important to define which mechanism(s)

are at play at these doses so that we can induce this type of response more consistently. Last, which of the many and diverse immunothera-pies (checkpoint blockade, therapeutic vaccines, positive co-stimulatory agonist abs, adoptive cell transfer, TLR agonists, among others) will ben-efit the most from the IM action of RT needs to be defined. Overall, such data will allow for determination of which CITs should routinely incorporate RT for its IM activities, and at what dose, allowing for the rationale incorporation of RT into CIT approaches.

Financial & competing interest disclosureC Garnett-Benson has been the recipient of an R21 from the NCI on radiation and T

REG cells (CA162235) and has a

pending Research Scholar Grant on radiation and immu-notherapy from the American Cancer Society. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Proofreading and editing services of Melissa Heffner of Second Look were utilized for the final manuscript produc-tion (funded by Georgia State University Department of Biology Internal Bridge Award).

EXECUTivE SUMMARYImmunomodulation by low-dose radiotherapy

● Low-dose radiotherapy (LD-RT) can modulate tumor phenotype.

● Immune cells can be directly modulated by LD-RT in ways that have been reported to be beneficial for antitumor efficacy.

● Single dose LD-RT inhibits NF-κB-mediated gene expression via proteasome inhibition.

● LD-RT and hypofractionated doses of RT appear to modulate NF-κB activity via different mechanisms in tumor cells.

Immunomodulation by hypofractionated doses of radiation

● Single dose RT (8–10 Gy) has been shown to modulate phenotype in vitro resulting in enhanced function of effector cytotoxic T lymphocytes (CTLs).

● Tumors irradiated with 10 Gy single dose (SD) enhance effector CTL viability and activity.

● Single dose RT in the hypofractionated dose range has been demonstrated to modulate tumor phenotype and gene expression by several diverse mechanisms.

● Antigen-presenting cells and TREGS exposed to SD radiation appear to be modulated in ways that could enhance the activities of cytotoxic immune cells.

Immunomodulation at work (functional outcomes)

● SD RT (8–10 Gy) has been shown to modulate phenotype in vivo and result in enhanced function of transferred CTLs and NK cells.

● 10 Gy SD can synergize with therapeutic cancer vaccines to induce an abscopal response.

● In vivo, RT has been shown to influence other noncytotoxic immune cells in a way that could positively influence antitumor attack by effector cells.

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ReferencesPapers of special note have been highlighted as: • of interest; •• of considerable interest

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84 Ling KL, Pratap SE, Bates GJ et al. Increased frequency of regulatory T cells in peripheral blood and tumour infiltrating lymphocytes in colorectal cancer patients. Cancer Immun. 7, 7 (2007).

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69Immunotherapy (2016) 8(1), 69–77 ISSN 1750-743X10.2217/imt.15.101 © 2016 Future Medicine Ltd

Immunotherapy

Review 2015/12/118

1

2016

Although treatment options for castration-resistant prostate cancer (CRPC) have increased over the last decade, there remains a need for strategies that can provide durable disease control and long-term benefit. Recently, immunotherapy has emerged as a viable and attractive strategy for the treatment of CRPC. To date, there are multiple strategies to target the immune system, and several approaches including therapeutic cancer vaccines and immune checkpoint inhibitors have been most successful in clinical trials. With regard to this, we report the results of the most recent clinical trials investigating immunotherapy in CRPC and discuss the future development of immunotherapy for CRPC, as well as the potential importance of biomarkers in the future progress of this field.

First draft submitted: 19 August 2015; Accepted for publication: 20 October 2015; Published online: 7 December 2015

Keywords: biomarker • cancer vaccine • immune checkpoint inhibitor • immunotherapy • prostate cancer

Prostate cancer remains highly prevalent and has a poor clinical outcome once metastatic. Localized prostate cancer can be managed with active surveillance, brachytherapy, exter-nal beam radiotherapy and radical prostatec-tomy with or without androgen deprivation therapy (ADT), depending on the individual prognosis [1]. Patients treated with ADT alone for metastatic disease usually respond well, but ultimately biochemical relapse and disease progression may occur. Disease that progresses despite ADT is referred to as cas-tration-resistant prostate cancer (CRPC) and most prostate cancer-related deaths occur in patients with metastatic CRPC (mCRPC). During the past 5 years, the US FDA has approved five new agents (sipuleucel-T, caba-zitaxel, abiraterone acetate, enzalutamide and radium-223) based on demonstrated overall survival (OS) benefit in Phase III studies [2–6].

Despite the improved therapeutic options for CRPC, there remains a need for treat-ments that can provide durable disease con-

trol and long-term survival benefit. Among the many strategies that are being investi-gated to address this need, immunotherapy is now an established treatment approach for prostate cancer with multiple clinical trials, such as randomized vaccination trials with sipuleucel-T and another with a recombinant virus-based vaccine, PROSTAVAC, demon-strating improvements in OS [7]. Although traditional treatment mostly focuses on androgen deprivation and chemotherapy, immunotherapy rely on activating the host’s immune cell to specifically attack prostate cancer cells and generate tumor-specific immunity. Based on this mechanism, immu-notherapeutic approach for prostate cancer may have broad applicability to all clinical states of the disease in the setting of neo-adjuvant, biochemical relapse post-primary therapy, castrate nonmetastatic and castrate metastatic. Improved understanding of the interactions between the immune system and prostate cancer has generated renewed

Immunotherapy in prostate cancer: challenges and opportunities

Masanori Noguchi*,1,3, Noriko Koga1, Fukuko Moriya2 & Kyogo Itoh3

1Division of Clinical Research, Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan 2Department of Pathology, Kurume University School of Medicine, Kurume, Japan 3Cancer Vaccine Center, Kurume University School of Medicine, Kurume, Japan *Author for correspondence: Tel.: +81 942 31 7989 Fax: +81 942 31 7866 [email protected]

part of

Review


70 Immunotherapy (2016) 8(1) future science group

Review Noguchi, Koga, Moriya & Itoh

interest in treating prostate cancer with immuno-therapy. This review presents the rationale for using immunotherapy to treat prostate cancer, summarizes recent clinical trial progress in particular CRPC, and discusses the future development of immunotherapy for patients with prostate cancer.

Principles of immunotherapy in prostate cancerThe primary aim of immunotherapy is to harness the immune system’s ability to recognize and destroy tumor cells. Cell types central to the recognition and destruction of tumor cells include macrophages, anti-gen-presenting cells (APCs), CD8+ cytotoxic T lymphocyte (CTL) cells and NK cells. Pathophysi-ologically, tumors are adept at developing pathways to suppress immune responses and escape from immune destruction, culminating in evasion and clinical pro-gression [8]. Potential mechanisms of evasion include the modulation of immune-inhibitory (checkpoint) pathways to suppress T-cell activity and the disruption of antigen processing and presentation [9]. Tumors can also recruit and promote the developing immunosup-pressive cells, such as Treg and myeloid-derived sup-pressor cells (MDSC) [10]. Tumors may directly or indirectly mediate the release of immunosuppressive factors, such as TGF-β and IL-10, which contribute to the development of an immunosuppressive micro-environment in and around the tumor [11,12]. In addi-tion, indole 2,3-dioxygenase, which can be expressed by dendritic cells, MDSCs and cancer cells, promotes the naïve T cells to Treg and increases IL-6 expres-sion with augmenting MDSC function [13]. Potential immunotherapeutic targets include these inhibitory factors in the tumor microenvironment, such as Treg, MDSC, IL-6 and indole 2,3-dioxygenase. Figure 1 shows the descriptive mechanism of immune responses in the tumor microenvironment.

Research suggests that prostate cancer is an immuno-logically modulated malignancy, and thus may be sensi-tive to immunotherapy. For example, data from studies that have evaluated the cellular composition of prostate tumors suggest that immune cell populations infil-trate the prostate gland [14,15]. Infiltrating leukocytes detected in prostate tumors include NK cells, effector cells and Treg cells, suggesting that both the innate and the adaptive branches of the immune system may play a role in mounting an attack against prostate can-cer cells [16]. In addition, prostate cancer is particularly well suited to immunotherapeutic approaches for three reasons [17]. First, the relatively slow growth pattern of prostate cancer allows time to develop an immune response. Second, the prostate is a highly differentiated, gender-specific organ and prostate cancer offers a vari-

ety of suitable antigen targets for cancer immunother-apy, such as prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), prostate-specific membrane antigen (PSMA) and prostate stem-cell antigen. Third, the use of PSA for the early detection of recurrent dis-ease allows for the initiation of vaccine immunotherapy while the tumor burden is still minimal.

Vaccine-based immunotherapyThe goal of vaccine-based immunotherapy is to stim-ulate a specific antitumor immune response against tumor antigens while minimizing collateral damage to normal tissues [11]. The five main types of vaccine-based immunotherapies studied in CRPC can be clas-sified as autologous, cell-based, viral-based, peptide-based and DNA vaccines. The important clinical trials of vaccine-based immunotherapy for CRPC are summarized in Table 1.

Sipuleucel-TSipuleucel-T is an autologous dendritic-cell vaccine prepared using the patient’s own peripheral blood mononuclear cells, obtained with leukapheresis and ex vivo incubated with a recombinant fusion protein consists of PAP and granulocyte-macrophage colony-stimulating factor (GM-CSF) [18,25]. Phase I/II clinical trials have shown that sipuleucel-T is well-tolerated and the patients develop appreciable antigen-specific T-cell responses and antibodies against the fusion protein after the treatment [19,20]. Three Phase III clinical trials have been completed and showed promising findings of this autologous vaccine. The first two studies com-pared patients with asymptomatic mCRPC assigned to placebo or sipuleucel-T. There was no difference in time to tumor progression, but there was a significant increase of median OS (25.9 vs 21.4 months and 19.0 vs 15.7 months) [26,27]. A third Phase III clinical trial known as the Immunotherapy for Prostate Adeno-carcinoma Treatment (IMPACT) trial showed a 4.1-month improvement in median OS [2], and this result led to the approval of sipuleucel-T as the first thera-peutic cancer vaccine by the FDA in 2010. These trials demonstrated that sipuleucel-T was well-tolerated and that most common adverse events were injection-site reactions, transient fever and flu-like symptoms. The biological activity was reported in a study of immune responses in prostate tumor tissue following neoadju-vant sipuleucel-T in patients with localized prostate cancer [28]. Prostatectomy specimens from vaccinated patients showed a greater than twofold increase in CD3+ and CD4+ T cells at the tumor interface. Similar results were also reported in a neoadjuvant peptide vac-cine study, in which prostatectomy specimens showed rapid infiltration of CD45RO+ activated/memory lym-

www.futuremedicine.com 71future science group

Immunotherapy in prostate cancer: challenges & opportunities Review

phocytes into tumor sites after the vaccination [21]. Ret-rospective analysis of the IMPACT trial suggested that sipuleucel-T has more clinical benefit in patients with a lower baseline PSA value than in patients with a higher baseline PSA value [22]. Patients with a lower baseline PSA had a median OS of 41.3 months with 13 months improvement than placebo, while patients with a higher baseline PSA had a median OS of 18.4 months with only 2.8 months improvement. These results demonstrate that a greater treatment benefit with sip-uleucel-T occurs in patients with earlier-stage disease and more favorable baseline prognostic factors.

GVAXGVAX is a cell-based vaccine derived from irradiated hormone-sensitive (LNCaP) and hormone-resistant (PC3) cell lines genetically modified to secrete GM-CSF. In a Phase I/II dose-escalating study of patients with metastatic prostate cancer, GVAX was associated with a tolerable safety profile, PSA decrease and stabi-lization, and a median OS time of 35.0 months with high-dose treatment [29]. These results prompted the initiation of two Phase III trials of CVAX. VITAL-1 randomized patients with asymptomatic mCRPC to GVAX or docetaxel-prednisone, while VITAL-2 ran-domized symptomatic patients to receive docetaxel or docetaxel plus GVAX. In the first Phase III study (VITAL-1), futility analysis indicated that the trial had a <30% chance of improved OS with GVAX, so

that trial was terminated [30]. In the second Phase III study (VITAL-2), a planned interim analysis demon-strated a trend toward more deaths in the GVAX arm and shorter median OS, leading to the trial’s closure as well [31]. Despite these results, GVAX continues to be explored in combination regimens and in other tumor types [32].

PROSTVAC (PSA-TRICOM)Viral vectors are attractive for use in cancer immu-notherapies as they can mimic natural infection and lead to the induction of immune response against the tumor antigen that they encode. PROSTVAC is a PSA-targeted poxvirus-based vaccine consist-ing of a heterologous prime-boost (vaccinia or fowl-pox virus vector) and three costimulatory molecules (TRICOM; B7.1, ICAM-1 and LFA-3) that serve to increase the PSA-specific immune response [33]. Sev-eral early trials showed that the treatment of PROS-TAVAC was well-tolerated with minimal toxicities (fever and injection-site reactions) [34–37]. In a mul-ticenter Phase II trial of PROSTAVAC, 125 patients with minimally symptomatic mCRPC were random-ized 2:1 to receive vaccine or placebo, respectively. As in the sipuleucel-T trials, there was no difference in terms of time to tumor progression. However, PROS-TAVAC resulted in an 8.5-month survival advantage (median OS: 25.1 months for the PROSTAVAC group vs 16.6 months for controls; p = 0.0061) and

Figure 1. Mechanism of immune responses in the tumor microenvironment.

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3-year survival of 30% for PROSTAVAC versus 17% for controls [38]. These trials suggested tumor-specific CTL responses and prolonged OS in patients treated with PROSTAVAC. A global Phase III ran-domized, placebo-controlled trial of PROSTAVAC (NCT01322490) is currently enrolling 1200 patients with asymptomatic or minimally symptomatic mCRPC, with OS as the primary endpoint. Patients are randomized to receive PROSTAVAC with GM-CSF, PROSTAVAC with placebo GM-CSF or double placebo. Data on the primary endpoint of OS are expected in December 2015.

Personalized peptide vaccinationVaccine antigens that are selected and administered without considering the host immune cell repertoires could not efficiently induce beneficial antitumor immune responses. In view of the complexity and diver-sity of the immunological characteristics of tumors and the immune cell repertoires of hosts, a new concept of personalized peptide vaccination (PPV) has been developed [39]. In this ‘personalized’ cancer vaccine formulation, appropriate peptide antigens for vaccina-tion are screened and up to four peptides are selected from a list of vaccine candidates in each patient, based on pre-existing host immunity. In several Phase I stud-ies of PPV for CRPC, PPV was well-tolerated with toxicities consisting mainly of injection-site reactions and cellular and humoral immune responses, and decreases in the PSA levels in some patients have been reported [23–24,40]. In a randomized, crossover, Phase II trial of PPV plus low-dose estramustine phosphate (EMP) comparing standard-dose EMP in HLA–A2+ or HLA–A24+ patients with CRPC, median progres-sion-free survival (PFS) was 8.5 months in the PPV group and 2.8 months in the EMP group (p = 0.0012), and the median OS for the PPV plus low-dose EMP group was not reached within 22.4 months, while the median OS for the standard-dose EMP group was 16.1 months (p = 0.033) [41]. These results suggest that this combination with a low-dose cytotoxic drug produces additional antitumor effects with minimum immunosuppression. In another Phase II study, the OS in docetaxel-resistant CRPC patients treated by PPV (n =20) was compared with that of historical control (n = 17) [42]. Median OS from the first day of progres-sive disease was 17.8 and 10.5 months in docetaxel-based chemotherapy-resistant CRPC patients receiving PPV and not receiving PPV, respectively (p = 0.166). These encouraging preliminary studies suggested that PPV warrants further study as a novel therapy for CRPC patients with progressive disease after docetaxel chemotherapy. A Phase III, randomized, placebo-con-trolled trial of PPV (UMIN000011308) is currently

enrolling 333 docetaxel-resistant CRPC patients, with OS as the primary endpoint. The results of this trial are eagerly anticipated.

DNA vaccineVaccines based on nucleic material from tumor-associ-ated antigens have also been studied in prostate cancer. For example, DNA vaccine pTVG-HP has produced immunological responses in patients with recurrent, localized prostate cancer, and some evidence of clini-cal responses [43]. Of eight patients who had a ≥200% increase in PSA doubling time, six had detectable long-term PAP-specific IFN-γ-secreting T-cell responses [44]. An ongoing Phase II trial (NCT00849121) evaluating the immunogenicity of pTVG-HP with GM-CSF as an adjuvant is ongoing.

Immune-checkpoint inhibitorImmune-checkpoint inhibitors have generated excite-ment recently because of their successful use in the treatment of multiple tumor types including meta-static melanoma. This newly developed class of agents interfere with the immune system’s autoregulatory mechanisms, thereby enhancing T-cell activity and potentiating antitumor effects using antibodies tar-geting immunological checkpoint regulators, such as CTL-associated antigen 4 (CTLA-4), programmed death (PD)-1 and its ligand (PDL-1), which down-regulate the immune response pathway [45]. This treatment strategy is considered to be a more active immunotherapeutic approach than vaccine-based immunotherapies.

Ipilimumab is a fully human monoclonal antibody that targets CTLA-4 to turn the immune response back on and augment T-cell-mediated immune responses [45]. Ipilimimub was approved by the FDA in 2011 for the treatment of advanced melanoma and is currently being investigated for the treatment of non-small-cell lung cancer, metastatic renal cell cancer and ovarian cancer. Regarding mCRPC, a Phase I/II report of a dose-escalating study of ipilimumab with and without radiation therapy to a single site in bone showed stable disease and several dramatic and dura-ble responses [46]. Common immune-related adverse events (irAEs) of ipilimimub in this study were diar-rhea (54%), colitis (32%) and pruritus (20%); grade 3/4 irAEs included colitis (16%) and hepatitis (10%). These results provided the impetus for a recently reported Phase III trial [47] for patients who progressed after docetaxel chemotherapy and were randomly assigned 1:1 to receive bone-directed radiotherapy fol-lowed by either ipilimumab at 10 mg/kg or placebo every 3 weeks for up to four doses. Nonprogressing patients could continue to receive ipilimumab or pla-



cebo as maintenance therapy every 3 months. There was no significant difference between the ipilimumab group and the placebo group (median OS: 11.2 vs 10.0 months; p = 0.053) in terms of OS as the primary endpoint. However, subgroup analyses suggested that ipilimumab might provide an OS benefit for patients with favorable prognostic features. For the subgroup of patients with an alkaline phosphatase <1.5-times the upper limit of normal, hemoglobin >11.0 mg/dl and no visceral metastases, median OS was 22.7 months with ipilimumab compared with 15.8 months with placebo (p = 0.0038).

Similarly, nivolumab (anti-PD-1) and anti-PDL-1 have shown robust activity in melanoma, renal cell, non-small-cell lung and bladder cancers, but minimal activity in prostate cancer [48,49]. The type of irAEs for nivolumab and anti-PDL-1 agents was similar to that seen with ipilimumab; dermatological adverse events, diarrhea and fatigue occurred in approxi-mately 20, 20 and 30% of cases, respectively. Despite these results, there is still focus on investigating their effectiveness in prostate cancer via other combination approaches [50].

BiomarkersIt would be important to identify predictive biomark-ers that could accurately assess antitumor immune responses and predict patient prognosis following the administration of cancer vaccines. Without clear markers to assess the clinical benefit in cancer vaccine treatments such as RECIST criteria or PSA decline, it might be difficult to integrate cancer vaccine treat-ments for prostate cancer [51]. In some clinical trials, several postvaccination biomarkers, including CTL responses, antibody responses, Th1 responses, delayed-type hypersensitivity and autoimmunity, have been reported to be associated with clinical responses [52–56]. Analysis of data with sipuleucel-T showed that markers of an antigen-specific immune response (APC num-bers, APC activation and total nucleated cell numbers) correlated with OS, demonstrating immune activation as potential biomarkers [57]. Another analysis of PPV in 500 advanced cancer patients also showed that both lymphocyte counts before vaccination and increased IgG response to the vaccinated peptides after vaccina-tion, along with performance status, were well corre-

lated with OS [53]. Other trial using ipilimumab and GVAX showed that high pretreatment frequencies of CD4+/CTLA-4+ T cell, CD4+/PD-1+ T cell or differ-entiated CD8+ T cells or low pretreatment frequencies of differentiated CD4+ T cell or Tregs were associated with significantly prolonged OS [58]. However, there are currently no validated biomarkers for cancer vac-cines in widespread use, and only survival benefit has been considered as a prominent endpoint in many cancer vaccine trials. Moreover, novel biomarkers for selecting patients who would benefit most from cancer vaccines remain to be addressed. Therefore, there is still an urgent need for definitive biomarkers to assess the benefits of immunotherapies.

Conclusion & future perspectiveImproved understanding of the interactions between the immune system and prostate cancer has gener-ated renewed interest in treating prostate cancer with immunotherapy. Immunotherapy will be most effec-tive when disease burden is a minimal stage disease. While there are several promising immunotherapeutic agents under study, sipuleucel-T is clinically available as the first in class antigen-specific autologous immu-notherapy approved for CRPC treatment. Ongoing Phase III trials of PROSTAVAC, PPV and the immune checkpoint inhibitor ipilimumab may soon broaden the scope of immunotherapies available to patients with CRPC. Current strategies are also exploring opti-mal combinations and sequencing of immunotherapies with other treatments. Moreover, novel biomarkers for selecting patients who would benefit most from immu-notherapies remain to be addressed.

Financial & competing interests disclosureNoguchi M has served as an advisory board consultant for

Green Peptide Co. Ltd. Itoh K has served as a consultant

and received research funding from Taiho Pharmaceutical

Company. Koga N and Moriya F declare no competing in-

terests. The authors have no other relevant affiliations or

financial involvement with any organization or entity with

a financial interest in or financial conflict with the subject

matter or materials discussed in the manuscript apart from

those disclosed.

No writing assistance was utilized in the production of this

manuscript.

Executive summary

• Immunotherapy has emerged as a viable and attractive strategy for the treatment of prostate cancer.• There are multiple ways to target the immune system.• Data from studies support the activity and safety of therapeutic cancer vaccines and immune checkpoint

inhibitors in prostate cancer.• No surrogate biomarkers for clinical outcomes in patients treated with immunotherapy have been identified.



ReferencesPapers of special note have been highlighted as: • of interest

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58 Santegoets SJ, Stam AG, Lougheed SM et al. T cell profiling reveals high CD4+CTLA-4 + T cell frequency as dominant predictor for survival after prostate GVAX/ipilimumab treatment. Cancer Immunol. Immunother. 62, 245–256 (2013).


Immunotherapy

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The poor prognosis of pancreatic cancer patients signifies a need for radically new therapeutic strategies. Tumor-targeted oncolytic viruses have emerged as attractive therapeutic candidates for cancer treatment due to their inherent ability to specifically target and lyse tumor cells as well as induce antitumor effects by multiple action mechanisms. Vaccinia virus has several inherent features that make it particularly suitable for use as an oncolytic agent. In this review, we will discuss the potential of vaccinia virus in the management of pancreatic cancer in light of our increased understanding of cellular and immunological mechanisms involved in the disease process as well as our extending knowledge in the biology of vaccinia virus.

Keywords: immunotherapy • oncolytic virus • pancreatic cancer • vaccinia virus

Pancreatic cancer remains one of the most difficult cancers to diagnose and treat. It is the fifth most common cause of cancer death in the UK with 1 and 5 years survival of 20.8 and 3.3%, respectively. These figures have hardly improved since the early 1970s [1]. Complete surgical resection remains the only curative treatment. Unfortu nately, less than 20% of pancreatic tumors are amenable to surgical excision at the time of diagnosis. However, even with complete surgical resection prognoses remains poor with 5 years survival around 20% [2,3]. Gemcitabine is the main chemotherapeu-tic agent approved for advanced pancreatic cancer. Despite being shown to improve life expectancy compared with 5-flurouracil, effect remains modest with median sur-vival around 6 months [4]. Combining gem-citabin therapy with erlotinib led to mini-mal increase in life expectancy from 5.9 to 6.2 months [5]. Therefore, new treatment strategies are clearly imperative.

Vaccinia virus (VV) has played a prominent role in one of the greatest achieve-ments in medical history: the eradication of smallpox (caused by Variola virus). Since

then, VV has been developed as a vector for vaccines against infectious diseases such HIV, influenza, malaria and tuberculosis as well as in immunotherapies [6] and oncolytic therapies for cancer [7,8]. With regards to the latter, the earliest studies, which mainly used replication attenuated VV recombinants for fear of toxicity, were relatively disappoint-ing in the clinic. Replication competent VVs retain their ability to lyse tumor cells and spread through tumor tissue. Recent advances in DNA recombinant technol-ogy enabling the rational manipulation of the viral backbone, coupled with the ever increasing knowledge gains in the fields of molecular virology and cancer cell biol-ogy have aided the development of safe and efficacious tumor-targeted oncolytic VVs. These are currently at the forefront of the most promising novel anticancer agents.

In this review, we will explore the potential of tumor-targeted oncolytic VV in the management of pancreatic cancer in light of our increased understanding of cellular and immunological mechanisms involved in the disease process as well as our extending knowledge in the biology of VV.

Vaccinia virus, a promising new therapeutic agent for pancreatic cancer

Chadwan Al Yaghchi1, Zhongxian Zhang2, Ghassan Alusi1, Nicholas R Lemoine1,2 & Yaohe Wang*,1,2

1Centre for Molecular Oncology, Barts

Cancer Institute, Queen Mary University

of London, UK 2National Centre for International

Research in Cell & Gene Therapy,

Sino-British Research Centre for

Molecular Oncology, Zhengzhou

University, China

*Author for correspondence:

Tel.: +44 207 882 3596

[email protected]

part of

Review



Review Al Yaghchi, Zhang, Alusi, Lemoine & Wang

Tumor-targeted oncolytic viruses as a new class of cancer therapeuticsTargeted therapy of cancer using oncolytic viruses (OV) has generated much interest over the past decades in the light of the limited efficacy and the sig-nificant side effects of standard cancer therapeutics for advanced disease [9]. OVs have become an increasingly popular anticancer therapy platform due to their abil-ity to selectively infect and lyse tumor cells (Figure 1). Cancer selectivity of OVs could be a result of natural tropism [10,11] or via genetic modification [9]. OVs can target multiple cellular pathways [12–14] minimizing the risk of tumor resistance and induce different modes of cell death [15–18]. In addition, OVs can break down the immunosuppressive tumor microenvironment and induce a long-lasting tumor-specific immunity [19,20] (Figure 2). OVs can specifically deliver therapeutic pro-teins into tumors at increasing levels following viral replication within the malignant cells. Furthermore, OVs can function in synergy with conventional can-cer treatments of chemoradiotherapy [21–24]. Finally, OVs as a treatment platform are amenable to adjust-ment and development following our ever-increasing understanding of cancer cells, the virus and host immune responses to both tumor and virus.

H101, an adenovirus with E1B 55K gene deletion (Oncorine; Shanghai Sunway Biotech, Shanghai, China) was licensed in China in 2005 as the world’s first OV for treatment of head and neck cancer when combined with chemotherapy [25]. The similar virus, dl1520 (also known as, ONYX-015) has been adminis-tered by intratumoral injection under CT guidance into locally advanced primary tumors of pancreatic cancer patients in Phase I/II trials. The treatments were well tolerated, but no objective responses were seen in any of the patients with virus alone, and only 10% (2/21) patients showed objective response when gemcitabine

was used in combination [26–28]. Another virus that entered clinical trials is HF10, a Herpes Simplex virus armed with granulocyte-macrophage colony-stimu-lating factor (GM-CSF). Phase I trial of intratumoral injection into nonresectable pancreatic tumors proved to be safe with some encouraging clinical results [29]. These early results warrant further investigation to seek more powerful agents for this cancer.

Favorable features of vaccinia virus for cancer treatmentVV is a member the poxvirus family. It is a double-stranded DNA virus ∼192 kbp in size. It can be sta-bly accommodate up to 25 kbp of cloned exogenous DNA [30]. Structurally, it consists of a core region composed of viral DNA and a various viral enzymes including RNA polymerase and polyA polymerase encased in a lipoprotein core membrane. The outer layer of the virus consists of double lipid membrane envelope [31,32]. VV has two major forms of infectious virions; the intracellular mature virions, as described above, which is released upon cell lysis and the extra-cellular enveloped virion released from the cells via cell membrane fusion. The latter has an additional lipid bilayer membrane wrapped around the intracellular mature virion particle.

VV has many inherent characteristics that make it an ideal choice for oncolytic virotherapy. VV has a short life cycle of 8 h that takes place in its entirety in the cytoplasm eliminating the risk of genome integra-tion. Replication usually starts 2 h after infection, at which time the host cell nucleic acid synthesis shuts down as all cellular resources are directed toward viral replication [33,34]. Cell lyses takes place between 12 and 48 h releasing packaged viral particles. Further-more, the virus does not depend on host mechanisms for mRNA transcription making it less susceptible to biological changes of the host cell [33,35].

Unlike other OVs, VV does not have a specific surface receptor for cell entry allowing it to infect a wide range of cells unhindered by the lack of expression of said receptor. They depend on a number of membrane fusion pathways for cell entry [36,37].

The existence of various antigenically distinct forms of the mature virus allows it to evade host immune sys-tem. extracellular enveloped virion form of the virus is encapsulated in a host-derived envelope, with incorpo-rated viral proteins, that contains several host comple-ment control proteins [38–40]. In addition, VV infected cells secret Vaccinia complement control protein which binds an inactivate C4b and C3B inhibiting the classic and alternative complement activation pathways [41–43]. VV therefore can be disseminated relatively unharmed in the blood stream to reach distant tumors allowing

Figure 1. Tumor selectivity of oncolytic viruses. Tumor-targeted oncolytic viruses can exploit defective cellular pathways in cancer cells (top). oncolytic viruses can infect and replicate in cancer cells leading to cell lysis and release of viral particles. These in turn infect neighbor tumor cells and so forth. In normal cells (bottom) cellular defense mechanisms prevents viral replications.

Oncolytic virus

Cancer cell

Normal cell


Vaccinia virus, a promising new therapeutic agent for pancreatic cancer Review

systemic delivery of the virus [44], which is more suitable for the treatment of the advanced pancreatic cancer.

The hypoxic nature of pancreatic cancer contributes to its aggressive and treatment-resistant phenotype. In contrast to adenovirus [45], we have found that hypoxic conditions did not affect replication, viral proteins pro-duction, cytotoxicity and transgene expression of the Lister strain of VV [46]. These results suggest that VV could be suitable for management of pancreatic cancers and potentially other hypoxic tumors.

Finally, VV has a good safety track record following its use as a vaccine for over a century. Minor and less severe side effects include fever, rash and inadvertent inoculation. Moderate-to-severe side effects include eeczema vaccinatum, generalized vaccinia, progres-sive vaccinia and postvaccinial encephalitis [47]. Sides effects are rare with an incident of less than 1:10,000 and severe side effects in particular are extremely rare [48]. Genetically modified recombinant VV could be potentially safer due to their tumor selectivity. Recent clinical trial of JX-594 virus in hepatocellular carcinoma showed the treatment to be well tolerated with mainly flu-like symptoms in all patients and a single severe side effect [8].

How Vaccinia virus selectively kills cancer cells by multiple action mechanismsVV has a natural tropism to cancer cells [49,50]. The virus can utilize activated molecular pathways in tumor cells to aid its replication [51–53]. In fact, many of the hallmarks of cancer [54] make tumor cells sus-ceptible to viral replication including immune escape, sustained cell proliferation and resisting cell death. In the case of VV, the EGFR family [55], potentially plays an important role in tumor selectivity. The viral SPGF, an EGF-like growth factor carried by VV, can activate host cellular pathways leading to increased viral rep-lication [56]. In addition, Ras–GTP-activating protein S3H domain-binding protein, overexpressed in most human cancers [57], plays a role in VV replication by complementing the activity of the VITF-2 [58].

Various approaches can be utilized to enhance tumor selectivity of OVs. The virus depends for its replication in normal cells on a set of genes that prepare the cell resources for viral replication and block apoptotic path-ways. Deleting these genes will limit the virus ability to replicate in normal cells. However, these pathways are often disrupted in cancer cells allowing the mutant virus to replicate despite the defective genes. One such example is the disruption of the vaccinia thymidine kinase gene (TK gene) affecting the virus ability to synthesize deoxyribonucleotides [59,60]. Normal cells have a much smaller reserve of deoxyribonucleotides,

compared with tumor cells, limiting the ability of VV to replicate. Another example is the deletion of the B18R gene encoding the secreted IFN-binding protein that blocks IFNα signaling [61]. In normal cells, this gene deletion attenuates viral replication due to IFN antiviral effect while cancer cells remain permis-sive to VV replication as IFN signaling is often dis-rupted [62,63]. In addition, altering the expression of crucial vaccinia viral gene by microRNA also enables tumor-specific viral replication, which is a potentially novel and versatile platform for engineering VVs for cancer virotherapy [64].

GLV-1h68 is a replication-competent VV targeted at tumor cells by mutation of J2R (encoding thymidine kinase) and A56R (encoding hemagglutinin) loci. This virus was shown to be effective against human pancreatic cancer cell line in vitro and in nude mice xenografts. Importantly this efficacy was enhanced

Figure 2. Multiple modes of actions of tumor-targeted oncolytic viruses. Oncolytic viruses (OV) can kill cancer cells via a variety of mechanisms. First, they directly infect, replicate and lyse tumor cells sparing normal cells. Released virions can infect neighbor tumor cells and so forth. Second, OVs can induce immunogenic cell death associated with the release of pathogen-associated molecular patterns and damage-associated molecular patterns. In addition viral infection results in the release of cytokine and chemokines deviating the immune response toward a cytotoxic profile. Dendritic cells can pick tumor-associated antigens released from lysed tumor cells and prime CD8+ T cells to induce a tumor-specific immune response. Third, OV infection can result in vascular shutdown caused by direct viral invasion of endothelial cells and thrombosis caused by cytokine-mediated neutrophils accumulation. CD8: Cytotoxic T cell; DC: Dendritic cell; EC: Endothelial cell; N: Neutrophil; TAA: Tumor-associated antigen; TC: Tumor cell; VV: Vaccinia virus.

Direct cell lysis

Anti-tumor immuneresponse

DC

T-cell printing

CD8

CTL-mediatedcytotoxicuty

TAAs

Cell entry and replication

VV

EC

Vascular shut down

Direct invasion ofendothelial cells

NThrombosis

TC



when virus therapy was combined with gemcitabine and cisplatin [65]. GLV-1h151, a virus with similar gene deletions but different marker proteins transgenes [66], was found to be effective in vivo and in vitro against human pancreatic cancer cell lines. Combining the virus with radiotherapy resulted in a synergistic antitumor effect [67].

In addition to direct cell lysis, VV can utilize vascular shut down to kill noninfected tumor cells [44,68–69]. This is believed to be caused by accumulation of neutro phils in blood vessels, mediated by cytokines and chemokines, leading to intravascular thrombosis [69]. In addition, VV can infect and destroy tumor-associ-ated endothelial cells further contributing to vascular collapse [62]. Although this process has not been spe-cifically shown in pancreatic tumors, we believe it to play an important role in the multimechanistic antitu-mor effect of VV, as pancreatic cancers are often well-vascularised and high microvascular density correlates with poor outcome after surgical excision [70]. To fur-ther capitalize on this process we have rationally armed Lister strain VV with endostatin–angiostaten fusion gene, a well-documented angiogenesis inhibitor [71]. The resultant VVhAE virus proved to be tumor selec-tive in vitro and in vivo. It resulted in suppression of angiogenesis and prolonged survival of mice bearing human pancreatic cancer xenografts [50].

Vaccinia virus as immunomodulatory agentThe ability of OVs to alter the immune composition of the, ordinarily, immune-suppressive tumor microenvi-ronment led to a new line of thinking of their mecha-nism of action. Large body of evidence suggests that antitumor immunity, where the virus is acting as an oncotropic immunomodulator, is the key determinant of a successful onclytic virotherapy [72–74].

VV kills cancer cells via a combination of necrosis and immunogenic apoptosis resulting in the release of damage associated molecular patterns [75–78] and pathogen associated molecular patterns [79–81] as well as the release of viral antigens into the tumor. This process leads to a strong inflammatory response that can overcome the immune suppression within the tumor microenvironment. In addition, tumor cell lysis releases tumor-associated antigens (TAA) into this inflammatory environment. Dendritic cells recruited by the virus can in turn pick up these exposed TAAs and cross-prime CD8+ T cells resulting in a potent antitumor adaptive immune response. It has been dem-onstrated that an oncolytic VV (JX549) could induce tumor-specific immunity in human cancer patients [82] and preclinical study [20]. Therefore, oncolytic viro-therapy may be considered as a method of vaccination in situ, enabling the adaptive immune response to clear

residual disease as well remote metastatic cancer cells and provide long-term surveillance against relapse.

In the context of vaccination, heterologous prime-boost immunization regimen using recombinant adenovirus prime and VV boost has been shown to enhance CD8+ T-cell immunogenicity with protective efficacy against malaria in a mouse model [83,84]. So, it seems logical that combining two different OVs for cancer treatment may induce a stronger tumor- specific immunity. We have, for the first time, combined the use of oncolytic adenovirus and VV, in a prime-boost strategy, for treatment of established tumors in the hope to harness the host immune response to the infected tumor cells. We found that sequential treat-ment via intratumoral injection with oncolytic adeno-virus followed by oncolytic VV resulted in complete eradication of subcutaneous pancreatic cancer grafts in Syrian hamsters. More importantly, the surviv-ing animals developed a long-lasting tumor-specific immune response that protected them against tumor rechallenge. This process was shown to be T-cell dependent [20].

Arming VV with various cytokines and chemokines can further enhance its antitumor activity. IL-10, a cytokine produced by Th2 T cells, is a potent inhibitor of antiviral immune response [85]. We have found that arming VV with IL-10 dampened antiviral immune response resulting in prolonged viral persistence in pancreatic tumors. This led to stronger antitumor immunity and improved survival in both subcutaneous and transgenic pancreatic cancer mouse models [86].

Vaccinia virus as vaccine vectorThe first use of a recombinant virus armed with an antigen from a different organism as a vaccine vec-tor was reported over 30 years ago. VV armed with hepatitis B surface antigen gene was able to induce a protective immunity against hepatitis in chimpan-zees [87,88]. Since then there has been a great progress in recombinant VV vaccines in the veterinary field [89,90]. Unfortunately this success did not extend to human infectious diseases vaccines, mainly due to the lengthy and more stringent process for human licensing, with only a handful of recombinant VV vectors in current clinical trials [91–94].

One of the significant challenges for cancer vaccina-tion lies in developing strategies to improve the delivery of antigens to antigen-presenting cells in vivo, allowing effective antigen processing and presentation and acti-vation of a potent immune response against a unique background of immune tolerance toward ‘self ’ TAAs. Viral vectors have become attractive antigen delivery systems as they mimic a natural viral infection, resulting in induction of cytokines and co-stimulatory molecules



that provide a powerful adjuvant effect and elicit potent cellular immunity [74,95].

Survivin is a member of the inhibitor of apoptosis family expressed in a variety of cancers. It plays a cru-cial role in tumor survival and drug resistance [96]. It is expressed during embryonic development but absent from differentiated cells [97]. Survivin is overexpressed in 70–80% of pancreatic cancers and is associated with resistance to chemoradiotherapy [98,99]. Vaccination with Vaccinia Ankara virus, a nonreplicating attenu-ated VV strain, armed with survivin induced survivin-specific CD8+ immune response resulting in a modest antitumor effect. When combined gemcitabine anti-tumor immunity and efficacy improved significantly. This is likely to be related to gemcitabine suppression of myeloid-derived suppressor cells [100].

The only VV-based cancer vaccine to enter clini-cal trials is PANVAC-V, a VV expressing carcino-embryonic antigen and mucin-1, both highly expressed in pancreatic cancers. The two antigens were packaged with three costimulatory molecules: B7.1 (cluster of differentiation 80), ICAM-1 (intracellular adhesion molecule one) and LFA-3 (leukocyte function-asso-ciated antigen-3) known collectively as TRICOM. To further enhance the immune response, the vac-cination was delivered as a heterologous prime/boost regimen using a nonreplicating fowlpox vector express-ing the same antigens and costimulatory molecules (PANVAC-F) [101]. GM-CSF was administered at the injection site as an adjuvant to enhance local antigen processing and presentation. In a Phase I clinical trial, the vaccine was found to be safe and well tolerable. It generated an antigen-specific immune response toward carcinoembryonic antigen and mucin-1 which corre-lated with increased survival [102]. However, Phase III trial (NCT00088660) targeting patients with meta-static pancreatic cancer who failed gemcitabine treatment failed to meet its therapeutic targets and was terminated [103]. The vaccine is currently under investigation for direct intratumoral injection under endoscopic ultrasound guidance with encouraging results of Phase I trial [104].

Future perspectiveThere has been a great interest in VV in recent years. Its safety, cancer tropism, amenability to genetic modification and ability to target solid tumors via a variety of mechanism of actions have made it a near-perfect onclytic virus to target pancreatic cancers. Nevertheless, as with any new therapeutic agents VV therapy need to overcome many hurdles and challenges before it enters routine clinical practice.

The first challenge is the selection of the right VV strain. The nonvaccine strain Western Reserve (WR)

VV is widely used in the lab. JX-963, a GM-CSF armed mutant of WR VV with deletion of both the Thymidine Kinase and the Viral Growth Factor gene, has been reported as the most potent tumor-targeted oncolytic VV [52]. Other strains, such as the European vaccine Lister strain, are largely untested. We recently evaluated the antitumor potency and biodistribu-tion of different VV strains using in vitro and in vivo models of cancer, including pancreatic cancer models. The Lister strain virus with Thymidine Kinase gene deletion (VVΔTK) demonstrated superior antitumor potency and cancer-selective replication in vitro and in vivo, compared with WRDD, especially in human cancer cell lines and immune-competent hosts. Fur-ther investigation of functional mechanisms revealed that Lister VVΔTK presented favorable viral biodistri-bution within the tumors, with lower levels of proin-flammatory cytokines compared with WRDD, sug-gesting that Lister strain may induce a diminished host inflammatory response [105]. Our comprehensive study indicates that the Lister strain VV with TK deletion is a particularly promising VV strain for the develop-ment of the next generation of tumor-targeted onco-lytic therapeutics. We anticipate that more and more people will use the Lister stain of VV as a backbone to develop new OVs for cancer treatment in the future.

Further genetic modifications of VV might enhance its oncolytic ability. Disruption of the N1L gene reduces virulence and inhibits VV replication in the brain reducing the risk postvaccinial encephalitis, a rare but significant complication of VV vaccina-tion [106,107]. Our unpublished work on N1L-deleted VV suggests that N1L-deleted VV resulted in a supe-rior antitumor efficacy compared with N1L-intact VV [Ahmed J et al., Unpublished Data]. In addition, arming the new generation of VV with immune-modulatory genes or other therapeutic genes that enhance the antitumor immunity is a future for cancer treatment using tumor-targeted OVs.

Achieving the right immune response is of a paramount importance. Increasingly safer viruses permits the use of higher doses to maximize thera-peutic effect [8], however higher viral load might devi-ate the immune response toward antiviral immunity resulting in rapid viral clearance and reduced anti-tumor immunity. Manipulating the immune system with cytokine-armed viruses is not without its risks including serious autoimmune side effects [108].

Systemic delivery of VV is particularly relevant in pancreatic cancer as most pancreatic tumors present with distant metastasis at the time of diagnosis. One such virus (JX594) has recently been shown to effec-tively target tumors after intravenous infusion, making it an ideal OV for treatment of inaccessible tumors such



as pancreatic cancer [7]. To date, the systemic delivery of OVs has been shown to be safe but not efficacious mainly due to the rapid clearance of these agents by the immune system [109]. When designing new strat-egy to enhance the systemic delivery of VV lessons can be learnt from other OVs. Serotype exchange [110,111], engineering new serotypes [112] and the use of chemical shielding [113] have been successfully used with other OVs. In fact the latter strategy have been used to modify the nonreplicating Vaccinia Ankara vaccine vector to circumvent pre-existing anti-VV immunity [114]. Other approaches include pharmacologically modifying the immune response to reduce the neutralization of the systemically delivered OVs [115–117].

Combining oncolytic virotherapy with traditional cancer treatments is an area of great promise. Gem-citabine can suppress myloid-derived suppressor cells in the tumor microenvironment resulting in a stron-ger antitumor immune response [118]. On the other hand, gemcitabine is a nucleoside analogue that inhib-its DNA synthesis including that of double-stranded DNA viruses [119]. Using these agents in a sequential rather than combination manner might be the key to effective therapy [120]. Similarly, combining OVs with immune checkpoints inhibitors is an area that requires more investigation and optimization. PD-1 and CTLA-4 inhibitors might enhance the OV-induced antitumor immunity by creating a favorable immune profile in the tumor microenvironment [121,122].

Despite the challenges, the field of oncolytic viro-therapy is generating a great interest of both researchers and pharmaceutical companies alike. The recent US FDA approval of talimogene laherparapvec (T-VEC, an engineered herpes simplex virus-1 expressing GM-CSF),

for the treatment of melanoma has given the field a much needed boost. As safety and efficacy data start to accu-mulate the process of licensing new OVs will get easier. We anticipate other cytokine- and chemokine-armed viruses to enter clinical practice within the next few years. In addition, combining OVs with immune check-point therapies, monoclonal antibodies and CAR-T ther-apies will be an area of major research interest in the near future. Combining immune checkpoint antibodies with other immune-stimulating agents such as conventional drugs, targeted agents and most of all OVs, may increase the tumor types and individual patient profiles in which a durable clinical benefit can be achieved. OVs are finally being recognized for their ability to stimulate antitumor immunity, and with anti-CTLA-4 and anti-PD-1 agents on the market, OVs may finally have met their perfect match. It has never been a more promising era for cancer immunotherapy and personalized medicine.

We believe at the current rate of development it will not be long before OVs are part of routine clinical practice.

Financial & competing interests disclosureThis project was funded by the Medical Research Council of

the UK (MR/MD15696/1), Ministry of Sciences and Technol-

ogy, China (2013DFG32080) and the UK Charity Pancreatic

Cancer Research Fund as well Pancreatic Cancer Research

UK. The authors have no other relevant affiliations or finan-

cial involvement with any organization or entity with a finan-

cial interest in or financial conflict with the subject matter

or materials discussed in the manuscript apart from those

disclosed.


manuscript.

Executive summary

• Pancreatic cancer is one of the most aggressive human cancers, without effective therapies.• Tumor-targeted oncolytic viruses is a new class of cancer therapeutic agents.• Oncolytic Vaccinia virus (VV) has distinctive features that make it ideal for treatment of pancreatic cancer.• The antitumor efficacy of oncolytic VV can be further improved by modification of viral genes and arming the

virus with therapeutic genes.• Combination of oncolytic VV with other cancer therapies could be the future for treatment of pancreatic

cancer.

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http://www.prnewswire.com/news-releases/therion-reports-results-of-phase-3-panvac-vf-trial-and-announces-plans-for-company-sale-56997582.html


Immunotherapy

Review 2015/11/307

12

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2015

The field of immunotherapy in urinary malignancy is expanding in several directions and checkpoint inhibitors are leading the way. The aim of this report is to highlight the efficacy and safety profile of the two classes of molecules,anti-cytotoxic T-lymphocyte antigen-4 and anti-programmed death receptor-1/programmed death ligand type 1, that are under investigation and represent potential candidates to be used in the near future for the management of bladder and renal cell cancer. The preliminary results as well as the future perspectives of this novel immunotherapy are analyzed. Novel immune checkpoint targets are reviewed as well.

First draft submitted: 6 July 2015; Accepted for publication: 22 September 2015; Published online: 23 November 2015

Keywords: bladder cancer • checkpoint inhibitors • CTLA4 • immunotherapy • PD1/PDL1 • renal cell carcinoma

A novel immune targeted therapy: innovation or renovation?The hallmarks of cancer are defined as acquired functional multistep capabilities allowing cancer cells to survive, proliferate and disseminate [1]. Genomic instability asso-ciated with the inflammatory microenviron-ment of premalignant and malignant lesions driven by cells of the immune system are the two main promoter of carcinogenesis [2–4]. An increasing body of evidence suggests that the immune system plays a pivotal role in resist-ing or eradicating formation and progression of cancer [5,6]. Cells and tissues are constantly under immune surveillance and cancers that do develop have been able to break the har-mony of normal checkpoints to escape detec-tion and elimination by the host immune system [7–9]. Historically, oncologic treatment consisted of local disease control either by sur-gery or radiation therapy associated or not with systemic chemotherapy and was conceived as the only modality to treat and eventually cure cancer. However, until the last decade most of the chemotherapeutic drugs used in cancer

therapy worked by killing normal and malig-nant cells without discrimination leading to severe side effects and thus, decreasing dra-matically the quality of life of survivors. The narrow margin between the efficacy and the toxicity of such therapies associated with the unsatisfactory cancer control rates has urged the researchers to multiply their effort in order to understand the molecular biology of can-cer [10]. This has resulted in the development of a new era of treatment based on targeting more specifically the tumor. Several biologic agents mimicking or blocking some of the natural signals used by the body to control cell growth and proliferation, induce apoptosis, inhibit angiogenesis and increase immunity were developed [10]. Unfortunately, clinical results for most of these targeted therapies were partially disappointing due to their limited efficacy with short lasting tumor responses in the majority of cases and a common non-neg-ligible toxicity profile. Therefore, more effec-tive and less toxic treatments were needed to be developed to ensure effective cancer control while maintaining patients’ quality of life.

Checkpoint inhibitors in bladder and renal cancers: results and perspectives

Fouad Aoun*,1, Hampig R Kourie2, Spyridon Sideris2, Thierry Roumeguère3, Roland van Velthoven1 & Thierry Gil21Department of Urology, Jules Bordet

Institute, 1 Héger Bordet Street,

1000 Brussels, Belgium 2Department of Oncology, Jules Bordet

Institute, 1 Héger Bordet Street,

1000 Brussels, Belgium 3Department of Urology, Erasme

Hospital, Route de Lennik 808,

1070 Brussels, Belgium


Tel.: +32 0032 2541 3125

Fax: +32 0032 2541 3987

[email protected]

part of

Review



Review Aoun, Kourie, Roumeguère, Velthoven & Gil

A new resource of knowledge on biological signaling machinery in cells, molecular patterns of tumors and pathways of interaction between immune host cells and tumor antigens confirmed the cancer fundamental biology and helped to elucidate the mechanism behind the development of resistance to treatment. Instead of targeting cancer cells to induce a passive antitumor activity, a new concept of targeting the immune cells to generate active antitumor immunity was created [11]. The aim of this approach is to activate antitumor immune responses in order to break tumor tolerance, and stimu-late the immune response of patients against their own tumor antigens. This paradigm shift opened up a new perspective in treating cancer; several new agents are currently tested, as single agents or in combination, in clinical trials with promising results as demonstrated by durable remissions obtained in some patients. Some of these agents are currently evaluated for urologic cancers within ongoing clinical trials and the preliminary results are encouraging. The aim of this report is to highlight the efficacy and safety profile of two classes of molecules, anti-cytotoxic T- lymphocyte antigen-4 (CTLA-4) and anti-programmed death receptor-1 (PD1)/programmed death ligand type 1 (PDL1), that are under investigation and represent potential candidates to be used in the near future for the management of bladder urothelial cancer (BUC) and renal cell cancer (RCC). Novel immune checkpoint targets were also reviewed and summarized.

Therefore, we performed in August 2015 a comprehensive literature search in the Pubmed and Cochrane databases to identify clinical and random-ized controlled trials. The following terms were used: bladder cancer in combination with CTLA-4, PD-1, PDL-1, MHC-II, HLA-G, B7-H1, B7-H3, B7-H4 and TIM3 and kidney cancer in combination with the same molecules. We also searched abstracts from the major oncology conferences during 2013–2015. We then referred to the electronic site https://clinicaltrials.gov/ to search for ongoing trials.

Checkpoint inhibitors: different targets & mechanisms of actionThe immune system plays a pivotal role in the prevention of cancerogenesis. It is well established that immunocompromised patients are at increased risk of malignancies. A functioning immune system is required to eradicate the majority of viral infection capable of inducing tumors and to prevent the establishment of an inflammatory environment favorable to cancerogenesis. It can also specifically recognize and eliminate tumor cells by the innate and adaptive immune system. The latter process is the first phase of the sequential concept of immune editing and is referred to as the elimination phase or what was previously known as tumor immune

surveillance. Tumor cells that are not eliminated are thought to enter a temporary state of equilibrium in which latent cancerous cells are controlled by the adaptive immunity. In response to permanent immune pressure, cancerous cells accumulate selective changes enabling them to resist, suppress or escape the antitu-mor immune response. During the escape phase, tumors are capable of proliferation and invasion. Any immune molecule capable of stabilizing the equilibrium state or the inhibition of tumor escape mechanism might play a crucial role in cancer control [12].

T-cell checkpoint inhibitors: dawn of a new age of managementT-cell checkpoints are normal immune signals expressed on lymphocytes [13]. Under normal physiologic condi-tions, these molecules are the inhibitory pathways that counterbalance the costimulatory pathways in order to limit permanent immune stimulation and to minimize collateral damage to normal tissues.

CTLA-4 was among the first of such molecules identified as a negative regulatory molecule expressed by activated T cells; it is related to the T-cell costimula-tory receptor CD28:B7 immunoglobulin superfamily that competes with CD28 for B7 and its interaction with CD 80/CD86 results in an inhibitory signal lead-ing to deactivation of tumor-specific T cells. Signal-ing through CD28 increases mRNA expression of the IL-2 and promotes T-cell survival and differentiation and immunoglobulin isotope switching. CTLA-4- mediated extracellular domain signals are therefore inhibitory and turn off T-cell-dependent immune responses [14]. Another mechanism involves the cyto-solic domain of CTLA-4 and occurs at lower levels of surface expression. The inhibitory actions of CTLA-4 are thought to occur via its interaction with the tyro-sine phosphatases. Another T-cell checkpoint inhibi-tor, PD-1, was similarly identified as a negative regu-lator of T-cell proliferation and functions in order to maintain peripheral immune tolerance [15]. The PD-1 gene is a CD28 family member that is a member of the immunoglobulin gene superfamily and is mainly expressed on activated T cells, B cells and myeloid cells. PD-L1 and 2 (PD-L2) are cell surface protein of B7 family member. PD-L1 is expressed on immune, nonhematopoietic and tumoral cells while PD-L2 is expressed primarily on macrophages and dendritic cells as well as on the normal parenchymal cells of lung and kidney. The PD-1 contains an intracellular proximal immune-receptor tyrosine inhibitory motif and distal immunoreceptor tyrosine-based switch motif. When activated, the receptor delivers a negative signal by the recruitment of a protein tyrosine phosphatase SHP-2 to the phosphorylated tyrosine residue in the immuno-

https://clinicaltrials.gov/


Checkpoint inhibitors in bladder & renal cancers Review

receptor tyrosine-based switch motif in its cytoplasmic region. The major role of PD-1 is to limit the activity of T cells in peripheral tissues during an inflammatory response to infection.

The apparent tolerance of the immune system, in tissues and within the tumor microenvironment, stems, at least partially, from the activation of checkpoint inhibitors allowing tumor cells to evade immunologic response [16,17]. In fact, these cells produce substantial amounts of costimulators of checkpoint inhibitors such as PD-L1 and PD-L2. It had been demonstrated that interaction between PD-1 expressed on activated T cells and PD-L1 expressed on antigen-presenting cells and tumor cells leads to phosphorylation of the intracellular portion of PD-1 and inhibition of T-cell receptor signal-ing. This will result in a downregulation of the activity of effector T cells and lead to death of activated T cells, limiting, therefore, the immunologic response within the tissues and tumor microenvironment [18]. Similarly, an inhibitory mechanism through the CTLA-4 pathway had been described.

Inhibiting PD1 or PDL1 by specific antibody could counteract the immune escape mechanism and ensure an adequate immunologic response in tissues and within the tumor microenvironment. Several anti-PD-1 (nivolumab, pembrolizumab, pidilizumab and AMP-224) and anti-PD-L1 antibodies (atezolizumab, MDX-1105, MEDI14736 and MSB0010718C) are currently inves-tigated. Among anti-PD-1 antibodies, nivolumab (MDX-1106) and pembrolizumab (MK-3475), fully human, IgG4 (kappa) isotype, monoclonal antibodies that binds PD-1 and blocks PD-L1 and PD-L2 from binding to PD-1 are the most advanced checkpoint inhibitors in the field of metastatic refractory RCC. For anti-PD-L1 antibodies, atezolizumab, also known as MPDL3280A, a high-affinity engineered human anti-PD-L1 monoclonal immunoglobulin-G1 antibody is the most investigated. It targets PD-L1 expressed on tumor cells and tumor-infiltrating immune cells, preventing it from binding to PD-1. In addition, PD-L1 exerts an inhibitory signal on T cells by interacting with B7 on the surface of T cells. Unlike anti-PD-1, anti-PD-L1 monoclonal antibodies will prevent this latter interac-tion, although the effect of this remains unknown. Another slight difference is that PD-1 interaction with PD-L2 provides unique negative signaling that prevents autoimmunity. Thus, anti-PD-1 antibody blockage of such an interaction may remove this inhibition, allowing autoimmune adverse effects. Anti-PD-L1 antibody, how-ever, would theoretically leave PD-1–PD-L2 interaction intact, preventing the autoimmunity caused by PD-L2 blockade. In terms of antitumor activity, both anti-PD-1 and anti-PD-L1 antibodies have shown comparable responses in various kinds of tumor.

Similarly, by blocking the CTLA-4 with specific antibodies, this previously described inhibition will be overcome and subsequently the tumor specific T cells will be activated, thereby leading to the migration of an increased number of activated T cells to the tumor site. At the tumor site, inhibition of CTLA-4 will elim-inate the regulatory T cells allowing activated T cells to attack and kill tumor cells [19]. Ipilimumab is a fully human monoclonal IgG1κ that binds to the CTLA-4 antigen expressed on a subset of T cells. The proposed mechanism of action for ipilimumab is its interference with the interaction of CTLA-4 with B7.1 molecules on dendritic cells, with subsequent blockade of the inhibitory modulation of T-cell activation promoted by the CTLA-4/B7 interaction. This will allow the cytotoxic T lymphocytes to destruct tumor cells [19].

T-cell checkpoint inhibitors in renal cell carcinoma: fixed concept & new eraHistorically, RCC and melanomas were the only two solid cancers considered to be immunologically influ-enced. These tumors presented objective responses and durable remissions, albeit in a small subset of patients when treated with immune therapies. As a remain-der, IL-2 was approved in 1992 by the US FDA as a treatment for metastatic RCC after 14% of partial and complete objective response rate (ORR) in these patients in a study published by Fyfe et al. [20]. Another cytokine IFNα-2a was approved as a first-line treat-ment in metastatic RCC with ORR varying between 6 and 12% [21,22]. Afterward, IFNα-2a in association with bevacizumab showed significantly longer interval of progression-free survival (PFS) and a trend toward longer overall survival (OS) compared with IFNα-2a alone in two randomized studies [23,24]. Based on these two studies, the combination of IFNα-2a and bevacizumab was approved by the EMA and the FDA for the first-line treatment of patients with advanced or metastatic RCC in 2007 and 2009, respectively.

In the new era of the checkpoint inhibitors, promising results are eagerly awaited for the treatment of RCC. More than ten ongoing clinical trials are eval-uating these drugs in the treatment of RCC as mono-therapy, or in association with antiangiogenesis drugs or as a combination of two checkpoint inhibitors, in metastatic disease or before surgical resection (Table 1).

Anti-CTLA-4: the first Phase I trial of ipilimumab only reported the incidence of hypophysitis and enterocolitis in a mixed cohort of patients treated for metastatic melanoma or RCC [25,26]. The efficacy of ipilimumab had been also evaluated as monotherapy in patients with stage IV RCC, who received IL-2 as first-line treatment or who are ineligible to receive this treatment [27]. Sixty-one patients were treated with a



Table 1. Ongoing trials using checkpoint inhibitors in renal cell carcinoma.

Study Drug Phase Indication Compared with Primary outcome

NCT02210117 Nivolumab Phase II, a pilot randomized presurgical study

Metastatic RCC/eligible for cytoreductive nephrectomy

Nivolumab + bevacizumab and nivolumab + ipilimumab

Safety and tolerability of nivolumab versus nivolumab + bevacizumab versus nivolumab + ipilimumab

NCT02212730 Pembrolizumab Phase I Patients undergoing surgical resection for RCC

NA Side effect and evaluation of intratumoral lymphocytic infiltration

NCT02348008 Pembrolizumab with bevacizumab

Phase Ib/II Metastatic RCC NA Maximum safe dose of treatment regimen and efficacy of treatment regimen

NCT01354431 Nivolumab Phase II randomized, blinded, dose-ranging study

Progressive, advanced/metastatic clear-cell RCC who have received prior antiangiogenic therapy

NA PFS in different arms (different doses)

NCT01668784 Nivolumab Phase III, randomized, open-label

Advanced or metastatic clear-cell RCC who have received prior antiangiogenic therapy

Everolimus OS (criteria met and the study was interrupted prematurely)

NCT01984242 Atezolizumab ± bevacizumab

Phase II, randomized study

Untreated advanced RCC

Sunitinib Safety/efficacy study PFS

NCT02089685 Pembrolizumab + PEG-IFN or ipilimumab

Phase I/II Advanced melanoma and advanced RCC

NA Safety, tolerability and PFS of these combinations

NCT02133742 Pembrolizumab and axitinib

Phase Ib, open label, dose finding study

Previously untreated advanced RCC

NA Number of participants with dose-limiting toxicities

NCT02231749 Nivolumab + ipilimumab

Phase 3, randomized, open-label study

Previously untreated, advanced or metastatic RCC

Sunitinib PFS and OS

NCT02014636 Pembrolizumab Phase I, II and III Treatment-naive subjects with advanced RCC

Pembrolizumab and pazopanib and pazopanib alone

Safety/efficacy study OS

NCT01472081 Nivolumab + sunitinib, ipilimumab or pazopanib

Phase I Metastatic RCC NA Safety and tolerability of these combinations

NCT00057889 Ipilimumab Phase II IL-2 refractory or IL-2 ineligible patients with Stage IV RCC

NA Efficacy

NA: Not applicable; OS: Overall survival; PFS: Progression-free survival; RCC: Renal cell cancer.



loading dose of 3 mg/kg followed by three doses of 1 mg/kg in 21 patients (group 1) and 3 mg/kg in the remaining 40 patients (group 2). Patients with stable disease, partial or complete responses were allowed additional treatment. Only one (5%) patient in the first group experienced partial response compared with five (12.5%) patients in the second group. The prob-ability of response increased to 21% in treatment-naive subjects. The duration of the response varied between 7 and 21 months. In another study, three doses of tremelimumab (6, 10, 15 mg/kg) were investigated in association with sunitinib. ORR for the combination was comparable to Phase III trials of sunitinib alone [28].

AntiPD-1/PD-L1: in a multicenter Phase I dose escalation trial, Brahmer et al. investigated BMS-936559, an anti-PD-L1 monoclonal antibody, in 207 advanced cancer patients (17 patients with advanced RCC). An objective response and a stable disease (at least 6 months) based on RECIST criteria were noted in two and seven patients with advanced RCC, respectively [29]. The same authors reported a 6-month PFS of 56% in another cohort of heavily treated RCC patients [30]. Cho et al. presented the preliminary results of atezolizumab show-ing 6 months PFS of 50% in patients with metastatic RCC [31]. The CA209–003 Phase I trial assessed the clinical activity of nivolumab in patients with advanced RCC [32]. Promising long term survival outcomes were achieved with nivolumab. The median OS was >22 months. Nivolumab was administered safely in the outpatient setting for up to 2 years. The study also evaluated treatment-related adverse effects. Adverse effects developed in 75% of patients. Grade 3 and 4 toxicities were encountered in 17% of patients. Select adverse effects occurring in ≤1% of patients included

lung infiltration (n = 3; 1.0%), acute respiratory distress syndrome (n = 1; 0.3%), acute respiratory failure (n = 1; 0.3%), diabetes mellitus (n = 1; 0.3%), hypoph-ysitis (n = 1; 0.3%), renal failure (n = 3; 1.0%), and tubule-interstitial nephritis (n = 2; 0.7%). There were three (1%) deaths in patients with pneumonitis-induced adverse events (two patients with non-small-cell lung cancer, one patient with RCC).

A randomized, dose ranging Phase II trial of nivolumab for metastatic clear cell RCC progressing after prior ther-apies and within 6 months of enrollment (CA209–010) were then undertaken [33]. The researchers randomized patients into three arms: patients in the first, second and third arms received 0.3, 2 and 10 mg/kg of nivolumab intravenously every 3 weeks, respectively. Treatment was continued until progression or intolerable adverse effects occurred. ORRs were similar and no dose–response relationship was observed for PFS across the three doses studied. Antitumor activity for nivolumab, including durable responses and promising OS outcomes (18.2, 25.5, 24.7 months in the first, second and third arm, respectively) were demonstrated in this pretreated popu-lation. These median OS are higher than those reported in other pretreated population of RCC Phase III trials (Table 2). It is noteworthy to mention that improved OS could well be due to the difference in the size and designs of these studies and not be a true marker of increased effi-cacy in this population. Of note, no patients experienced a Grade 4 or 5 treatment-related events. Grade 3 toxicities occurred in 11% of patients and were manageable.

In a Phase Ia study investigating atezolizumab in metastatic RCC patients, the drug was well toler-ated with no treatment-related deaths [34]. Evidence of increased activity was observed in patients with

Study Drug Phase Indication Compared with Primary outcome

NCT01441765 CT-011 Phase II Stage IV RCC PD-1 with dendritic cell/renal cell carcinoma Fusion cell vaccine

Safety

NCT02446860 Nivolumab Phase I/II Metastatic RCC with no prior systemic therapy and scheduled to undergo nephrectomy

Safety response rate, PFS, OS, changes in biomarkers, changes in biomarkers correlated with both response to treatment and toxicity

NCT02348008 Pembrolizumab Phase I/II Metastatic RCC Pembrolizumab with bevacizumab

Safety and efficacy

NA: Not applicable; OS: Overall survival; PFS: Progression-free survival; RCC: Renal cell cancer.

Table 1. Ongoing trials using checkpoint inhibitors in renal cell carcinoma (cont.).



elevated PD-L1. Durable responses (54 weeks) and prolonged stable disease were observed. The 24-week PFS rate was 53%. Pembrolizumab (MK-3475) is tested in a Phase I trial in patients undergoing surgical resection for RCC (NCT02212730).

Combination therapy: Nivolumab evaluated in patients with metastatic RCC, who were eligible for cytoreductive surgery, was compared with nivolumab in combination with bevacizumab or ipilimumab (NCT02210117). The latter combination is appealing for the concept of maximum blockade and for coun-teracting immune resistance [35]. Results of these com-parisons are awaited. However, the CA209–016 Phase I trial that evaluated the safety of the combination of nivolumab and ipilimumab had reported recently their results [36]. In this study, patients were randomized into two arms: patients in the first arm received four dos-ages of nivolumab 3 mg/kg and ipilimumab 1 mg/kg iv. every 3 weeks and patients in the second arm received four dosages of nivolumab 1 mg/kg and ipilimumab 3 mg/kg iv. every 3 weeks. After the completion of the four schedules, patients continued nivolumab 3 mg/kg iv. every 2 weeks. Antitumor activity of the combination was demonstrated in this study as only 24 and 13% of patients had a progressive disease, in the first and second arm, respectively. The 6-month PFS rates were similar in the two arms (65 vs 64%; p < 0.05) and higher than reported previously with nivolumab or ipilimumab monotherapy in RCC. Responses were also durable even after discontinuation of study drugs. All patients in the second arm developed treatment-related side effects with 60% having grade 3 or 4 leading to discontinu-ation of therapy in 26% of patients. The combination was better tolerated in the first arm but severe adverse effects occurred in 9.5% of patients. On the basis of

this study, a Phase III randomized, open-label study comparing nivolumab combined with ipilimumab (the same schedule of patients of the first arm in the previous study) to sunitinib monotherapy (50 mg/day for 4 weeks followed by 2 weeks off) in subjects with previously untreated, advanced or metastatic RCC with primary outcome PFS and OS is ongoing (NCT02231749).

Nivolumab (MDX-1106) was also evaluated as monotherapy in a Phase II trial to determine the PFS and Phase III trial (compared with everolimus) to determine as a primary end point OS in patients with advanced or metastatic RCC who have received prior antiangiogenic therapy (NCT01668784). The latter trial randomized 821 patients to receive either nivolumab 3 mg/kg intra-venously every 2 weeks or everolimus 10 mg daily and was interrupted prematurely because the primary end point was met demonstrating superior OS in patients in the nivolumab arm compared with the everolimus arm. This drug was also used in Phase I trial in combination with sunitinib, pazopanib and ipilimumab in metastatic RCC to evaluate the safety and tolerability of these com-binations (NCT01472081). A trial investigating the combination of nivolumab with sunitinib or pazopanib is ongoing. Preliminary results revealed comparable 50% ORRs [37]. Atezolizumab and pembrolizumab are being tested in combination with bevacizumab or VEGF-targeted drugs and in comparison to VEGF-targeted drugs in untreated patients with metastatic RCC (NCT01984242).

Checkpoint inhibitors in bladder cancer: evolving therapeutic landscape for advanced & metastatic patientsUntil the mid-1980s, life expectancy for patients with advanced or metastatic BUC was about 6 months [38]. The

Table 2. Comparison of overall survival between CA209–010 Phase II trial and targeted therapies Phase III trials.

Axis INTORSECT RECORD-1 GOLD CA209–010

Drug tested Axitinib Temsirolimus Everolimus Dovitinib Nivolumab(0.3, 2, 10 mg/kg)Sorafenib Sorafenib Placebo Sorafenib

Number of patients 389 512 416 570 168

Risk group

Favorable NS 19 29 20 33

Intermediate NS 69 56 58 42

Poor NS 12 14 22 25

Prior therapy/line of therapy

Sunitinib/2nd Sunitinib/2nd VEGF/2nd or higher

VEGF and mTOR/3rd or higher

VEGF and/or mTOR/2nd and higher

Median OS (CI 95%) 15.2 (12.8–18.3) 12.3 (10.1–14.8) 14.8† 11.1 (9.5–13.4) 18.2 (16.2–24.0)

16.5 (13.7–19.2) 16.6 (13.6–18.7) 14.4† 11.0 (8.6–13.5) 25.5 (19.8–28.8)

24.7 (15.3–26.0)†Data not available.



modern era in the management of these patients started when two landmark cisplatin-based chemotherapy studies set the tone for future management of these tumors [39,40]. In the same period, endovesical bacillus Calmette–Guérin (BCG) instillation had supplanted cystectomy as the treatment of choice for carcinoma in situ (CIS) [41]. After 30 years of research, these chemotherapy regimens are still standard of care as first-line treatment for advanced or metastatic BUC. Numerous trials had evaluated new targeted therapies in this indication, but none showed better activity and/or efficacy compared with standard therapy. In addition, the mechanism of action of BCG is incompletely understood [42]. However, the efficacy of BCG in eradicating CIS and reducing the risk of recurrences in patients with nonmuscle inva-sive BUC opened the door to the development of novel immunotherapy agents for BUC [43]. Once in the blad-der, the BCG organisms enter macrophages and BUC cells and are broken down. Fragments are combined with histocompatibility antigens and displayed on the cell sur-face inducing cytokine and direct cell-to-cell cytotoxicity responses, which targets these cells for destruction. This had contributed to consider BUC as an immunologi-cally influenced malignancy. Afterward, two interesting immunologic hypotheses were emitted with potential therapeutic impact. The first one is based on the fact that immune system will consider the BUC cells as foreign, because of the high rates of somatic mutations in these cells [44]. Recently, a case–control study showed a signifi-cant association between CTLA-4 polymorphism and BUC risk [45]. Carthon et al. conducted the first preoper-ative clinical trial with ipilimumab (3–10 mg/kg for two cycles) as a neoadjuvant drug in 12 patients with muscle invasive BUC [46]. They demonstrated that ipilimumab has an acceptable safety profile before radical cystectomy. Giving immunotherapy preoperatively will provide a real-time evaluation of tumor response allowing discon-tinuation of ineffective therapies. The second immuno-logic hypothesis is that the cancer cells of the bladder will counteract the immune system through the expression of PDL1 in their microenvironment [47]. Therefore, several researchers examined the anti-PD1 and anti-PD-L1 anti-body for the treatment of BUC in different indications: as monotherapy in metastatic BUC or in combination with BCG in high-risk non-muscle invasive BUC, in first line or after failure of platinum-containing chemother-apy (Table 3). A Phase I study is evaluating anti-PDL1 in combination with BCG in high-risk non-muscle invasive BUC that has had removal of their bladder tumor, with safety as primary outcome (NCT02324582). Two Phase II trials using anti-PD1 and anti-PDL1 in metastatic BUC are launched; each trial will include more than 350 patients with primary outcome overall response rate (NCT02108652, NCT02335424). Two large Phase III

trials are evaluating anti-PD1 and anti-PDL1 as second-line treatment compared with vinflunine, paclitaxel or docetaxel in patients with metastatic BUC with OS as primary outcome (NCT02302807, NCT02256436). One trial is testing ipilimumab in association with gem-citabine-cisplatin in a Phase II trial as first-line treatment in patients with metastatic urothelial carcinoma with primary outcome 1-year OS (NCT01524991). All these trials are summarized in Table 3. Two trials had reported recently their results. Plimack et al. reported a response rate of 24% in 33 heavily treated patients with advanced urothelial cancer [48]. Powles et al. reported the result of a Phase I escalation and expansion study PCD4989g designed to evaluate as a secondary end point, the anti-tumor activity of atezolizumab in patients with locally advanced or metastatic malignancies [49]. They demon-strated that the drug had an antitumor activity in meta-static BUC with responses often occurring at the time of the first response assessment (6 weeks). A response rate of 43% was achieved in patients with tumors express-ing PD-L1-positive tumor-infiltrating immune cells. Of note, 11% of patients with negative PD-L1 expres-sion responded as well. The drug was well tolerated and no renal toxicities were reported. If the results of this study are replicated in ongoing Phase III trials, atezoli-zumab will be a great gain in the armamentarium of BUC therapies especially for elderly and/or patients with impaired kidney function.

Other immune checkpoint moleculesSeveral other checkpoint inhibitor molecules are under development and clinical evaluation. HLA-G, a HLA class I molecule that protects FETAL semi-allogeneic graft from maternal recognition, is gaining interest. The molecule is normally expressed at very low levels by adult tissues. By contrast, cancerous cells express on their cell surface HLA-G at different stages of their carcinogenesis process [50]. A soluble form was also isolated from the circulation of a humanized mouse model. It has been hypothesized that HLA-G had a broader immune-regu-lating function than CTLA-4 and PD-1. Its expression in tissue or as a soluble form is associated with higher tumor grade, tumor metastasis and poor survival in ani-mal models [51]. It plays a crucial role in the equilibrium phase and constitutes a novel immune escape mecha-nism. Anti-HLA-G antibodies restore in animal models antitumor immunity against HLA-G expressing tumor cells [51]. By examining the renal tissues obtained from 38 cases of RCC and in ten samples of normal kidney parenchyma, Kren et al. demonstrated a downregulation of HLA-G expression in almost all samples of normal kidney parenchyma and its upregulation in cancerous cells [52]. However, HLA-G expression was associated with better PFS rates. HLA-G is overexpressed in



Tab

le 3

. An

ti-P

D1/

PDL1

an

d a

nti

-CTL

A4

on

go

ing

tri

als

in b

lad

der

can

cer.

Stu

dy

Dru

gPh

ase

Ind

icat

ion

Co

mp

ared

wit

hPr

imar

y o

utc

om

e

NC

T021

08

652

Ate

zoliz

um

abPh

ase

II, m

ult

icen

ter,

n

on

ran

do

miz

ed, s

ing

le

arm

Loca

lly a

dva

nce

d o

r m

etas

tati

c B

UC

(ch

emo

ther

apy

nai

ve o

r p

rog

ress

ing

aft

er c

hem

oth

erap

y)N

AEf

fica

cy s

tud

y

OR

R

NC

T023

028

07A

tezo

lizu

mab

Phas

eIII

, op

en-

lab

el, m

ult

i cen

ter,

ra

nd

om

ized

Loca

lly a

dva

nce

d o

r m

etas

tati

c B

UC

aft

er f

ailu

re

wit

h p

lati

nu

m-c

on

tain

ing

ch

emo

ther

apy

Vin

flu

nin

e, p

aclit

axel

o

r d

oce

taxe

lSa

fety

/effi

cacy

stu

dy

OS

NC

T023

2458

2Pe

mb

roliz

um

ab

wit

h B

CG

Phas

e I

Hig

h-r

isk

no

n-m

usc

le in

vasi

ve B

UC

wh

o h

ave

had

re

mo

val o

f th

eir

bla

dd

er t

um

or

NA

Safe

ty (

gra

de

and

q

uan

tity

of

adve

rse

even

ts)

NC

T023

3542

4Pe

mb

roliz

um

abPh

ase

IIFi

rst-

line

trea

tmen

t o

f p

arti

cip

ants

wit

h a

dva

nce

d/

un

rese

ctab

le (

ino

per

able

) o

r m

etas

tati

c B

UC

wh

o

are

inel

igib

le f

or

cisp

lati

n-b

ased

th

erap

y

NA

Effi

cacy

stu

dy

OR

R

NC

T022

5643

6Pe

mb

roliz

um

abPh

ase

III R

and

om

ized

tr

ial

Rec

urr

ent

or

pro

gre

ssiv

e m

etas

tati

c B

UC

Vin

flu

nin

e, p

aclit

axel

o

r d

oce

taxe

lEf

fica

cy s

tud

y

OS

and

PFS

NC

T025

001

21Pe

mb

roliz

um

abPh

ase

II, r

and

om

ized

Aft

er fi

rst-

line

chem

oth

erap

y in

pat

ien

ts w

ith

m

etas

tati

c B

UC

Plac

ebo

Safe

ty/e

ffica

cy s

tud

y

PFS

NC

T018

48

834

Pem

bro

lizu

mab

Phas

e Ib

N

on

ran

do

miz

edU

roth

elia

l tra

ct c

ance

r o

f th

e re

nal

pel

vis,

ure

ter,

b

lad

der

or

ure

thra

NA

Safe

ty/e

ffica

cy s

tud

y

Ad

vers

e ev

ents

NC

T015

2499

1Ip

ilim

um

ab w

ith

g

emci

tab

ine

/ci

spla

tin

Phas

e II

Firs

t-lin

e tr

eatm

ent

for

pat

ien

ts w

ith

met

asta

tic

BU

CN

ASa

fety

/effi

cacy

stu

dy

1-ye

ar O

S

BC

G: B

acill

us

Cal

met

te–G

uér

in; B

UC

: Bla

dd

er u

roth

elia

l can

cer;

NA

: Not

ap

plic

able

; OR

R: O

bje

ctiv

e re

spo

nse

rate

; OS:

Ove

rall

surv

ival

; PFS

: Pro

gre

ssio

n-f

ree

surv

ival

.



urothelial cancer cells and anti-HLA-G antibodies are investigated in vitro and in vivo studies.

B7-H3 belongs to the B7 family of immune-regulatory molecules physiologically expressed by activated T cells, B cells, macrophages, dendritic cells and natural killer cells as well the liver, lung, breast, placenta and prostate. The role of B7-H3 in cancer progression remains con-tentious. In vitro and in vivo studies of the function of B7-H3 revealed both costimulatory and coinhibitory immune-regulatory functions on cellular and antitumor immune response. However, recent evidence suggests that nonimmunological effects play a major role in can-cer progression [53,54]. B7-H4 is another member of the B7 family recently identified as an inhibitory modulator of T-cell response. Ovarian tumor cells and melanoma cell lines were shown to overexpress B7-H4 protein in intracellular compartment. B7-H3 is mainly expressed in blood vessels surrounding RCC and its expression was associated with higher tumor grade, tumor metastasis and worse prognosis [55,56]. Similar findings have been suggested for B7-H4 expression [57]. Aberrant B7-H3 expression was also noted in BUC cells.

Lymphocyte activation gene 3 is a cell surface receptor that binds HLA class II molecules. It is expressed mainly on tumor cells, tumor infiltrating cells and macrophages. It inhibits natural killer and stimulates the inhibitory function of regulatory T cells. A synergic tumor escape function of PD-1 and LAG-3 was demonstrated in mice [58]. The safety of a recom-binant soluble LAG-3 fusion protein (0.050 to 30 mg/injection biweekly for six injections) was demonstrated in 21 patients with advanced RCC in a Phase I trial [59]. No clinically significant adverse events were observed. Tumor growth was reduced and PFS was better in those patients receiving higher doses compared with lower doses (87.5 versus 27.2%; p = 0.015).

T-cell immunoglobulin mucin-3 (Tim-3) is a mem-ber of immunoglobulin superfamily, physiologically detected on various immune cells. Tim-3 can regulate T-cell immune responses by accelerating the death of IFN-γ-producing type 1 helper T cells through interaction with its ligand, galectin-9 [60]. It has been suggested that Tim-3 plays a pivotal role in tumor-associated immune suppression and Tim-3 overexpres-sion has been reported in several human malignan-cies. Recently, the levels of TIM-3 were significantly increased on tumor infiltrating lymphocytes of 30 RCC patients [61]. High levels of expression were asso-ciated with higher stages of the disease. Similarly, aberrant TIM-3 expression was noted in cancer cells, tumor infiltrating lymphocytes and endothelial cells from 100 patients with BUC [62]. The levels of Tim-3 were significantly correlated with higher stage and grade. The authors also demonstrated that PD-1 was

over expressed on cancer cells and correlates well with TIM-3 expression. Of note, the expression of each molecule was an independent predictor of PFS and OS in these patients.

Conclusion & future perspective In terms of immunotherapy, we are currently in an immunotherapy revolution in oncology and checkpoint inhibitors will probably be the backbone of these therapies in the near future. They have already been approved as valid therapeutic options in melanoma, breast cancer and lung cancer providing a long-lasting disease control and a broad clinical activity [63,64]. In urologic cancer, it seems that we are witnessing the ‘dawn of a new age of man-agement.’ Oncologic results with nivolumab and atezoli-zumab in metastatic clear cell RCC are promising with long-lasting remissions in some patients. The median OS was >22 months with nivolumab [32]. At 1 and 2 years, survival rates were 70 and 50%, respectively. These rates were higher than those obtained with the same molecule in melanoma and non-small-cell lung cancer. Durable responses and tumor regression persistent after treat-ment discontinuation were noted in a subset of patients. Recently, the randomized Phase III trial comparing nivolumab to everolimus in VEGF refractory disease was stopped earlier because its primary end point – that is improved OS – was met in the arm receiving nivolumab. The breakthrough of its results is currently changing reimbursement policy and clinical practice. Preliminary data on the combination of anti-PD-1 or anti-PD-L1 with anti-CTLA-4 or VEGF-targeted therapy had showed higher PFS rates than those reported previously with monotherapy in RCC [65]. The concept of early intervention with VEGF-targeted therapy combined to immune checkpoint inhibitors is appealing. Multiple ongoing randomized first-line trials are testing this com-bination with primary end point improvement of initial control of the disease and long-lasting remissions. If posi-tive, these studies will change the treatment landscape of RCC. For BUC, atezolizumab and pembrolizumab have shown encouraging results in heavily pretreated chemo-therapy refractory metastatic patients. These drugs are currently investigated in chemotherapy-naive patients and results are eagerly awaited.

Of note, responses are variable with subsets of patients experiencing modest ORRs and PFS esti-mates. This highlights the concept of tumor hetero-geneity and urges the need for adequate biomarkers to evolve toward a more personalized medicine. Cur-rently, several biomarkers are examined to assess the immunomodulatory activity of checkpoint inhibitors (T-cell receptor sequencing, soluble factors, HLA-G, HLA-E, gene expression profiling…) and specific predictive biomarkers for a response are still lacking.



The level of expression of PD-1 on T cells, for example, was not considered a positive biomarker to treat the patients with an anti-PD1; patients without PD-1 expression have beneficiate, to a lesser extent from anti-PD-1 therapy [32]. The role of PD-L1 biomarker is rele-vant in both RCC and BUC but its identification varies substantially according to the assay used for measur-ing. Expression of circulating immune parameters and cancer cells neoantigens are under investigation and may represent the next biomarkers in the future. Opti-mizing immunohistochemical assays and identifica-tion of these molecules should continue to be explored. Of all the efficacy markers of drug activity retained, data are most promising for the duration of response. In the absence of a predictive biomarker, patient selec-tion based on medical history, presenting symptoms, tumor burden and prior therapies should be taken into account in the design of future studies to unravel the best sequence or combination of available therapies that will provide the greatest chance for transformative responses. Defining the mechanism of resistance to these drugs remains a challenge for modern oncology.

Finally, adverse effects should be addressed to clearly inform the patient before treatment. Safety of these drugs in urologic cancers was demonstrated as a primary end point in almost every Phase I/II trials. In general, these drugs were well tolerated but patients and clinicians should be aware that drug-related adverse effects could occur and that any organ system could be affected. The most frequent adverse effects are immune-mediated and include rash, vitiligo, diarrhea, colitis, hepatitis, pneumonitis, hypophysitis and fatigue [65]. However, most of the reported adverse effects were Grade 1/2 or

Grade 3 toxicities and were manageable with appropriate treatment, including high-dose corticosteroids and immunosuppressant. The overall rates of grade 4 toxic-ity varied between 0 and 17% among studies. Grade 5 toxicity has also been reported in some studies [27]. Pre-liminary data indicate that these side effects may be latent and appear or last longer than therapy [33]. It has been hypothesized that immune-related adverse effects of anti-CTLA-4 antibodies and their severity may cor-relate with objective tumor responses. Of note, no direct comparison of adverse effects between anti-CTLA-4, anti PD-1 and anti-PD-L1 antibodies had been made. Analysis of the different studies suggests that the type of immune-related adverse events was similar between the different agents. However, these adverse effects are more common and of higher grade with anti-CTLA-4 anti-bodies. Slightly higher rates of infusion reactions (11%) were observed with anti-PD-L1 compared with anti PD-1. The level of PD-L1 expression in tumor tissue may correlate with the severity of toxicity. A special attention should be paid to the safety and tolerability of nivolumab combined with ipilimumab.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial

involvement with any organization or entity with a financial

interest in or financial conflict with the subject matter or

materials discussed in the manuscript. This includes employ-

ment, consultancies, honoraria, stock ownership or options,

expert testimony, grants or patents received or pending, or

royalties.


manuscript.

Executive summary

• A new generation of immune therapeutics based on T-cell immune checkpoint inhibitors is gaining considerable attention in urologic oncology.

• Anti-cytotoxic T-lymphocyte antigen-4, anti-programmed death receptor-1 and anti-programmed death-ligand type 1 monoclonal antibodies have been investigated in urologic cancer.

• Preliminary oncologic results of all these classes of immune checkpoint inhibitors are promising.• Results of nivolumab and atezolizumab have been encouraging in metastatic renal cell cancer with long-

lasting responses, albeit in a small subset of patients.• Nivolumab improved overall survival of patients with VEGFR refractory renal cell cancer compared with

everolimus in a randomized Phase III trial.• Preliminary data on the combination of anti-programmed death receptor-1 or anti-anti-programmed death-

ligand type 1 with anti-cytotoxic T-lymphocyte antigen-4 or VEGF-targeted therapy had showed higher progression-free survival rates than those reported previously with monotherapy in renal cell cancer.

• Atezolizumab and pembrolizumab have shown encouraging results in heavily pretreated chemotherapy refractory metastatic patients.

• Adequate biomarkers are eagerly awaited in order to evolve toward a more personalized medicine.• Defining the mechanism of resistance to these drugs remains a challenge for modern oncology.• The safety and tolerability of these drugs were demonstrated as most of the reported adverse effects were

mild and manageable with appropriate treatment. However, the safety of the combination (nivolumab + ipilimumab) should be carefully explored.



ReferencesPapers of special note have been highlighted as: • of interest; •• of considerable interest.


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•• Averygoodreviewtounderstandthethreephases(elimination,equilibriumandescape)oftheconceptofimmunoeditingthatisthemostplausibletheorytoexplainhowcancercellsescapeimmuneresponse.

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through reduction of intratumoral regulatory T cells. Cancer Immunol. Res. 1(1), 32–42 (2013).

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19 Pentcheva-Hoang T, Simpson TR, Montalvo-Ortiz W, Allison JP. Cytotoxic T lymphocyte antigen-4 blockade enhances antitumor immunity by stimulating melanoma-specific T-cell motility. Cancer Immunol. Res. 2(10), 970–980 (2014).

20 Fyfe G, Fisher RI, Rosenberg SA et al.Results of treatment of 255 patients with metastatic renal cell carcinoma who received high-dose recombinant interleukin-2 therapy. J. Clin. Oncol. 13(3), 688–696 (1995).

21 Negrier S, Escudier B, Lasset C et al. Recombinant human interleukin-2, recombinant human interferon alfa-2a, or both in metastatic renal-cell carcinoma. Groupe Francais d’Immunotherapie. N. Engl. J. Med. 338(18), 1272–1278 (1998).

22 Motzer RJ, Hutson TE, Tomczak P et al. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N. Engl. J. Med. 356(2), 115–124 (2007).

23 Escudier B, Bellmunt J, Negrier S et al. Phase III trial of bevacizumab plus interferon alfa-2a in patients with metastatic renal cell carcinoma (AVOREN): final analysis of overall survival. J. Clin. Oncol. 28(13), 2144–2150 (2010).

24 Rini BI, Halabi S, Rosenberg JE et al. Bevacizumab plus interferon alfa compared with interferon alfa monotherapy in patients with metastatic renal cell carcinoma: CALGB 90206. J. Clin. Oncol. 26(33), 5422–5428 (2008).

25 Blansfield JA, Beck KE, Tran K et al. Cytotoxic T-lymphocyte-associated antigen-4 blockage can induce autoimmune hypophysitis in patients with metastatic melanoma and renal cancer. J. Immunother. 28(6), 593–598 (2005).

26 Beck KE, Blansfield JA, Tran KQ et al. Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4. J. Clin. Oncol. 24(15), 2283–2289 (2006).

27 Yang JC, Hughes M, Kammula U et al. Ipilimumab (anti-CTLA4 antibody) causes regression of metastatic renal cell cancer associated with enteritis and hypophysitis. J. Immunother. 30(8), 825–830 (2007).

28 Rini BI, Stein M, Shannon P et al. Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma. Cancer 117(4), 758–767 (2011).

29 Brahmer JR, Tykodi SS, Chow LQ et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366(26), 2455–2465 (2012).

30 Brahmer JR, Drake CG, Wollner I et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J. Clin. Oncol. 28(19), 3167–3175 (2010).

31 Cho BC, Sosman JA, Sznol M et al. Clinical activity, safety, and biomarkers of 552 MPDL3280A, an engineered PD-L1



antibody inpatients with metastatic renal cell 553 carcinoma (mRCC). J. Clin. Oncol. 31, Abstract 4505 (2013).

32 Mcdermott DF, Drake CG, Sznol M et al. Survival, durable response, and long-term safety in patients with previously treated advanced renal cell carcinoma receiving nivolumab. J. Clin. Oncol. 33(18), 2013–2020 (2015).

•• ReportedtheefficacyandsafetyofnivolumabinpatientswithadvancedVEGFrefractoryrenalcellcancer.Thestudydemonstratedimpressiveoverallsurvivalof22.4monthsanddurableresponsesthatinsomeresponderspersistedafterdrugdiscontinuation.Inaddition,thedrugwaswelltoleratedandtoxicitywasmanageable.

33 Motzer RJ, Rini BI, Mcdermott DF et al. nivolumab for metastatic renal cell carcinoma: results of a randomized Phase II trial. J. Clin. Oncol. 33(13), 1430–1437 (2015).

•• AmaturePhaseIIstudydemonstratingoverallsurvivalratesobtainedwithnivolumabhigherthanthoseobtainedwithVEGF-targeteddrugsinPhaseIIItrials.

34 Mcdermott DF, Sznol M, Sosman JA et al. Immune correlates and long term follow 567 up of a Phase Ia study of MPDL3280A, an engineered PD-L1 antibody, in patients with 568 metastatic renal cell carcinoma. Ann. Oncol. (S4), Abstract 8090 (2014).

•• Thefirststudyreportingthesafety(notreatment-relateddeaths)andefficacyofatezolizumab(24-weekprogression-freesurvivalratewas53%)inmetastaticrenalcellcancerpatients.Evidenceofincreasedactivitywasobservedinpatientswithelevatedprogrammeddeath-ligand1(PD-L1).

35 Duraiswamy J, Kaluza KM, Freeman GJ, Coukos G. Dual blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell rejection function in tumors. Cancer Res. 73(12), 3591–3603 (2013).

36 Hammers HJ, Plimack ER, Infante JR et al. Phase I study of nivolumab in 576 combination with ipilimumab in metastatic renal cell carcinoma. J. Clin. Oncol. 32(35S), 577 Abstract 4504 (2014).

37 Amin A, Plimack ER, Infante JR et al. Nivolumab in combination with sunitinib or 579 pazopanib in patients with metastatic renal cellcarcinoma. Ann. Oncol. 25(Suppl. 24), 580, iv362–iv583 (2014).

38 Babaian RJ, Johnson DE, Llamas L, Ayala AG. Metastases from transitional cell carcinoma of urinary bladder. Urology 16(2), 142–144 (1980).

39 Harker WG, Meyers FJ, Freiha FS et al. Cisplatin, methotrexate, and vinblastine (CMV): an effective chemotherapy regimen for metastatic transitional cell carcinoma of the urinary tract. A Northern California Oncology Group study. J. Clin. Oncol. 3(11), 1463–1470 (1985).

40 Sternberg CN, Yagoda A, Scher HI et al. Preliminary results of M-VAC (methotrexate, vinblastine, doxorubicin and cisplatin) for transitional cell carcinoma of the urothelium. J. Urol. 133(3), 403–407 (1985).

41 Shelley MD, Court JB, Kynaston H et al. Intravesical Bacillus Calmette-Guerin in Ta and T1 bladder cancer. Cochrane Database Syst. Rev. doi:10.1002/14651858 (2000) (Epub ahead of print).

42 Inamoto T, Azuma H. Immunotherapy of genitourinary malignancies. J. Oncol. 2012, 397267 (2012).

43 Wu Y, Enting D, Rudman S, Chowdhury S. Immunotherapy for urothelial cancer: from BCG to checkpoint inhibitors and beyond. Expert Rev. Anticancer Ther. 15(5), 509–523 (2015).

44 Cancer Genome Atlas Research Network. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature 507(7492), 315–322 (2014).

45 Wang L, Su G, Zhao X et al. Association between the cytotoxic T-lymphocyte antigen 4 +49A/G polymorphism and bladder cancer risk. Tumour Biol. 35(2), 1139–1142 (2014).

46 Carthon BC, Wolchok JD, Yuan J et al. Preoperative CTLA-4 blockade: tolerability and immune monitoring in the setting of a presurgical clinical trial. Clin. Cancer Res. 16(10), 2861–2871 (2010).

47 Chen DS, Irving BA, Hodi FS. Molecular pathways: next-generation immunotherapy – inhibiting programmed death-ligand 1 and programmed death-1. Clin. Cancer Res. 18(24), 6580–6587 (2012).

48 Plimack ER, Gupta S, Bellmunt J et al. Phase 1B study of pembrolizumab (pembro; MK-3475) in patients (pts) with advanced urothelial tract cancer. Ann. Oncol. 25(LBA23A) (2014).

•• Demonstratedpromisingresultsforpembrolizumabinheavilypretreatedadvancedurothelialcancerpatients.

49 Powles T, Eder JP, Fine GD et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 515(7528), 558–562 (2014).

•• Atezolizumabachieved43%responseratesinPD-L1-positivepatientsand11%inPD-L1-negativepatients.

50 Rouas-Freiss N, Moreau P, Lemaoult J, Carosella ED. The dual role of HLA-G in cancer. J. Immunol. Res. 2014, 359748 (2014).

51 Lin A, Zhang X, Xu HH et al. HLA-G expression is associated with metastasis and poor survival in the Balb/c nu/nu murine tumor model with ovarian cancer. Int. J. Cancer 131(1), 150–157 (2012).

52 Kren L, Valkovsky I, Dolezel J et al. HLA-G and HLA-E specific mRNAs connote opposite prognostic significance in renal cell carcinoma. Diagn. Pathol. 7(58), 58–65 (2012).

53 Brunner A, Hinterholzer S, Riss P et al. Immunoexpression of B7-H3 in endometrial cancer: relation to tumor T-cell infiltration and prognosis. Gynecol. Oncol. 124(1), 105–111 (2012).

54 Chen YW, Tekle C, Fodstad O. The immunoregulatory protein human B7H3 is a tumor-associated antigen that regulates tumor cell migration and invasion. Curr. Cancer Drug Targets 8(5), 404–413 (2008).

55 Qin X, Zhang H, Ye D et al. B7-H3 is a new cancer-specific endothelial marker in clear cell renal cell carcinoma. Onco. Targets Ther. 6 1667–1673 (2013).

56 Crispen PL, Sheinin Y, Roth TJ et al. Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma. Clin. Cancer Res. 14(16), 5150–5157 (2008).



57 Krambeck AE, Dong H, Thompson RH et al. Survivin and b7-h1 are collaborative predictors of survival and represent potential therapeutic targets for patients with renal cell carcinoma. Clin. Cancer Res. 13(6), 1749–1756 (2007).

58 Norde WJ, Hobo W, Van Der Voort R, Dolstra H. Coinhibitory molecules in hematologic malignancies: targets for therapeutic intervention. Blood 120(4), 728–736 (2012).

59 Brignone C, Escudier B, Grygar C et al. A Phase I pharmacokinetic and biological correlative study of IMP321, a novel MHC class II agonist, in patients with advanced renal cell carcinoma. Clin. Cancer Res. 15(19), 6225–6231 (2009).

60 Ngiow SF, Teng MW, Smyth MJ. Prospects for TIM3-targeted antitumor immunotherapy. Cancer Res. 71(21), 6567–6571 (2011).

61 Cai C, Xu YF, Wu ZJ et al. Tim-3 expression represents dysfunctional tumor infiltrating T cells in renal cell carcinoma. World J. Urol. doi:10.1007/s00345–015–1656–1657 (2015) (Epub ahead of print).

62 Yang M, Yu Q, Liu J et al. T-cell immunoglobulin mucin-3 expression in bladder urothelial carcinoma: clinicopathologic correlations and association with survival. J. Surg. Oncol. doi:10.1002/jso.24012 (2015) (Epub ahead of print).

63 Brahmer J, Reckamp KL, Baas P et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N. Engl. J. Med. 373(2), 123–135 (2015).

64 Weber JS, D’angelo SP, Minor D et al. Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 treatment (CheckMate 037): a randomised, controlled, open-label, Phase 3 trial. Lancet Oncol. 16(4), 375–384 (2015).

65 Gunturi A, Mcdermott DF. Nivolumab for the treatment of cancer. Expert Opin. Investing. Drugs 24(2), 253–260 (2015).

• Agoodreviewonthesafetyofnivolumabforthetreatmentofcancer.

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Manipulation of co-stimulatory or co-inhibitory checkpoint proteins allows for the reversal of tumor-induced T-cell anergy observed in cancer. The field has gained credence given success with CTLA-4 and PD-1 inhibitors. These molecules include immunoglobulin family members and the B7 subfamily as well as the TNF receptor family members. PD-L1 inhibitors and LAG-3 inhibitors have progressed through clinical trials. Other B7 family members have shown promise in preclinical models. TNFR superfamily members have shown variable success in preclinical and clinical studies. As clinical investigation in tumor immunology gains momentum, the next stage becomes learning how to combine checkpoint inhibitors and agonists with each other as well as with traditional chemotherapeutic agents.

Keywords: B7 family • checkpoint proteins • immunotherapy • TNFR superfamily • translational medicine

One of the hallmarks of cancer is the abil-ity of the malignant cell to escape eradication by the immune system [1]. Proposed over a century ago, the concept of immune con-trol of cancer continues to develop [2,3]. The existence of tumor antigens led Burnett and Thomas to form their hypothesis about can-cer immune surveillance where the adaptive immune system was responsible for prevent-ing the development of cancer in immuno-competent hosts. This hypothesis fell out of favor until the 1990s when improved mouse models of immunodeficiency were developed and particularly when the role of IFN-γ in promoting immune-mediated rejection of transplanted tumor in mice [4].

Tumors are variably infiltrated by cyto-toxic T lymphocytes (CTLs), but a dense infiltration portends a better prognosis [5–7]. The T-cell response follows a complex inter-action between an antigen-presenting cell (APC) and a T cell. TCR recognition of an antigen on MHC molecule is not suffi-cient, a second signal provided by a member of the B7 family is required [8]. CD28 pro-vides the primary co-stimulatory signal for

the activation of T cells after it engages B7-1 (CD80) or B7-2 (CD86) [9]. CTLA-4 is a CD28 homologue that interacts with B7-1 and B7-2 and, in contrast to CD28, provides an inhibitory signal [10,11]. Newly identified members of the B7 family also provide inhib-itory signals the roles of which continue to be explored [12]. Blocking CTLA-4 mediated inhibition of the T-cell effector response has been an attractive therapeutic target. Mono-clonal antibodies (mAb) that block CTLA-4 are effective in mouse models of a variety of tumors [13–15]. Ipilumumab (Yervoy®) is US FDA approved for the treatment of meta-static malignant melanoma and represents the first success story of T-cell checkpoint inhibitor immunotherapy [16].

A more recent success story in cancer immunology is that of PD-1. PD-1 was first identified in lymphoid cells lines induced to undergo programmed cell death [17]. Later reports noted that PD-1 is expressed on acti-vated T and B cells, dendritic cells (DCs) and monocytes upon stimulation where it is found to play an inhibitory role [18–20]. PD-1 is highly expressed on T cells and leads to

Emerging targets in cancer immunotherapy: beyond CTLA-4 and PD-1

Amer Assal1, Justin Kaner2, Gopichand Pendurti3 & Xingxing Zang*,2,4

1Department of Medicine, Adult Bone

Marrow Transplant Service, Memorial

Sloan Kettering Cancer Center, New

York, NY 10065, USA 2Department of Medicine, Montefiore

Medical Center, Bronx, NY 10467, USA 3Division of Hematology/Oncology,

Department of Medicine, Jacobi Medical

Center, Bronx, NY 10461, USA 4Department of Microbiology

& Immunology, Albert Einstein College of

Medicine, Bronx, NY 10461, USA


[email protected]



Review Assal, Kaner, Pendurti & Zang

T cell exhaustion [21,22]. PD-1 expression is also noted on CD4+ Foxp3+ regulatory T cells (Tregs) where it contributes to their inhibitory role [23]. Several mAbs targeting PD-1 have progressed through clinical tri-als. The first FDA approved mAb was pembroli-zumab (Keytruda®), also known as lambrolizumab after showing response rates in melanoma patients who have progressed after first line therapy including immunotherapy with ipilimumab (KEYNOTE-001 trial) or in comparison to investigator-choice chemo-therapy (KEYNOTE-002) [24,25]. The second PD-1 targeting mAb to receive FDA approval was nivolumab (Opdivo®). It was well tolerated in Phase I studies in solid tumors as well as lymphomas [26–30]. Similar to pembrolizumab, nivolumab was shown to be superior to chemotherapy in the second line setting in mela-noma in the Phase III CheckMate-037 trial [31,32]. Nivolumab yielded better survival and higher response rated in comparison to docetaxel in the treatment of advanced squamous non-small-cell lung cancer [33]. Other PD-1 mAbs include pidilizumab (CT-011) which was the first one to reach clinical trials and remains in development (studies reviewed in [34]) and more recently MEDI0680 which is entering clinical trials [35,36].

Blocking CTLA-4 and PD-1 are not the exclu-sive path toward T-cell ‘dis-inhibition’. A variety of immunomodulatory pathways have been studied and exploited clinically with varying degrees of success and are at different stages of clinical development. Other members of the B7 family, part of the immunoglobulin superfamily, include B7x, HHLA2 and B7-H3 which play an inhibitory role. VISTA, Tim-3 and LAG-3 are members of the immunoglobulin superfamily have also been shown to play an inhibitory role. Immunomodu-latory pathways include members of the TNF receptor family and their ligands which have been studied as targets for cancer immunotherapy. These inhibitory and stimulatory molecules that have been studied as therapeutic targets are depicted in Figure 1A & B, respec-tively. Finally, indoleamine 2,3–dioxygenase 1 inhibi-tors have also been studied as antitumor therapies as discussed below.

PD-L1 & PD-L2The first reported ligand for PD-1 is PD-L1 (B7-H1) with wide expression at the mRNA level in lymphoid and nonlymphoid tissues [37]. It is a cell surface protein that is expressed on activated APC, T and B lympho-cytes and other cells. It inhibits TCR mediated T-cell proliferation and cytokine production through the engagement of PD-1 [38]. The PD-1/PD-L1 interac-tion induce T-cell tolerance in lymphoid tissue before their exit to the periphery, and blockade of this interac-

tion can reverse T-cell anergy [39]. Additionally, PD-L1 expressed on tumor cells can also act as a ligand to deliver an anti-apoptotic signal that leads to resistance to cytolytic function of CTL as well as to Fas-induced and drug-induced apoptosis [40]. Another interesting fact is that B7-1 was also shown to interact with PD-L1 which results in inhibition of T cells [41,42]. A second ligand for PD-1 is PD-L2 (B7-DC), which inhibits TCR medi-ated T-cell proliferation and cytokine production [43,44]. It is mainly expressed on DCs and macrophages [44,45]. Recently, a novel binding partner for PD-L2 is identified named RGMb, which is important for the development of respiratory immune tolerance [46].

PD-L1 is expressed in a variety of human carcinoma specimens as well as hematological malignancies such as multiple myeloma, leukemia and peripheral T-cell lymphoma and has been correlated to poor progno-sis [8,34]. Several mAbs that target PD-L1 have reached clinical trials. BMS–936559 is a fully human monoclo-nal IgG4 antibody that blocks PD-L1 [47]. Anti-PD-L1 antibodies inhibited tumor growth in murine synge-neic tumor models with a durable antitumor immunity. BMS–936559 can reverse in vitro Treg mediated sup-pression and does not cause antibody-dependent cyto-toxicity or complement-dependent cytotoxicity [47]. The first clinical trial with BMS–936559 also dem-onstrated high tolerability and durable responses [47]. Other monoclonal anti-PD-L1 antibodies include MEDI4736 [48,49], atezolizumab (MPDL3280A) which demonstrated a 43% response rate in a Phase I clinical trial in metastatic urothelial bladder cancer patients resulting in an FDA breakthrough designation [50], and MSB0010718C which exhibits antitumor activ-ity by blocking PD-L1 as well as antibody-dependent cell-mediated cytotoxicity [51,52].

B7x (B7-H4/B7- S1)B7x is an inhibitory transmembrane protein that binds activated T cells and is a member of the B7 fam-ily [53–55]. It inhibits CD4 and CD8 cell proliferation and cytokine production [53]. It is hardly expressed on professional APC but is expressed on nonlymphoid tis-sues, mainly epithelial tissues where a role in immune tolerance is postulated [54,56–58]. It is expressed in the lung epithelium and is implicated in attenuating the immune response to bacterial infection in mice [56]. B7x is expressed in a variety of human cancers which include cancers of the brain, esophagus, lung, breast, pancreas, kidney, gut, skin, ovary and prostate [59]. Prostate cancer specimens from patients treated with radical prostatectomy had 15% prevalence of B7x expression and high expression was significantly asso-ciated with a higher risk of prostate cancer related death [60]. B7x expression in renal cell carcinoma is

www.futuremedicine.com 1171

Figure 1. Summary representation of T-cell molecules. (A) Summary representation of T-cell co-stimulatory molecules. (B) Summary representation of T-cell co-inhibitory molecules.

Immune cells or tumor cells

MHC

MHC

CTLA-4PD-L1

PD

-L1

PD

-L2

HHLA2 B7x

PD-1Tim-3BTLA

T cells

T cells

TCR

TCR

Co-stimulation

Co-inhibition

T-cell proliferation and effector function

T-cell proliferation and effector function

?

? ? ? ?

TMIGD2

HHLA2B7-H3

B7-H3 VISTA HV

EM

Gal

-9

HM

GB

1

CE

AC

AM

1

GITRL

GITR

CD70CD40

CD40L CD27

OX40L

OX40

CD137L

CD137

ICOSL

ICO

SL

ICOS

B7-1 B7-2

B7-1 B7-2

CD28

Immune cells or tumor cells

Immunotherapy © Future Science Group (2015)


Emerging targets in cancer immunotherapy: beyond CTLA-4 & PD-1 Review

associated with adverse clinical and pathological fea-tures as well as poor survival [61]. Tumor expression of B7x in human gastric cancer predicts poor survival [62]. Similar findings were also reported in studies of ovarian cancer and lung cancer [63,64]. In a preclinical model, mouse colon carcinoma cells line CT26 transfected with murine or human B7x resulted in a higher number of lung metastasis and shorter survival [65]. Blockade of B7x with a mAb resulted in a reduction of number of lung metastasis in a CT26 as well as 4T1 based mouse models of lung metastasis [65]. B7x thus represents a very promising target for cancer immunotherapy.

HHLA2 (B7y/B7-H5/B7H7)HHLA2 is another member of the B7 family that mod-ulates T-cell function [66,67]. It is expressed on mono-

cytes and induced on CD19 positive B cells. HHLA2–Ig fusion protein bound resting and activated CD4 and CD8 T cells, as well as APC. It was shown to inhibit proliferation of CD4 and CD8 T cells in the presence of TCR signaling as well as T-cell cytokine produc-tion [66]. TMIGD2, also called CD28H or IGPR–1, is identified as one of the receptors for HHLA2 [67,68]. IGPR–1 was initially reported to be an adhesion mol-ecule involved in angiogenesis [68]. HHLA2 expression in non-lymphoid tissues was limited to placenta, GI tract, kidney, gallbladder and breast, but its expres-sion was more common in human tumor specimens including breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney and esoph-agus [68]. In a cohort of 50 patients with triple negative breast cancer, 56% of patients had HHLA2 expression



on their tumors, and high HHLA2 expression was sig-nificantly associated with regional lymph node metas-tasis and stage. Of interest, increase in HHLA2 expres-sion was also due to an increase in gene copy number, and not just stimulation [68]. There is much to be dis-covered about HHLA2 and it represents a potential target for cancer immunotherapy.

B7-H3B7-H3 was first identified as a molecule that binds a receptor on activated T lymphocytes [69]. Its expression was inducible on DCs and was initially thought to be costimulatory to T lymphocytes [69]. In vivo studies in mouse models showed that B7-H3 was an inhibitory to T lymphocytes and preferentially inhibits T helper cells type 1 response [70]. The receptor for this ligand is still unclear. It is expressed in some cancer cells and was associated with regional nodal metastasis [12,71]. Currently the majority of evidence suggests that this is a co-inhibitory ligand for T-cell response [72]. B7-H3 was found to be upregulated in graft-versus-host dis-ease (GVHD) target organs and its absence in B7-H3-/- mice resulted in augmented GVHD lethality and T-cell proliferation and function [73]. Increased B7-H3 expression in cancer specimens has been reported [74], and has been correlated to worse outcomes [60,75]. Therefore, B7-H3 is another potential target for cancer immunotherapy.

VISTAVISTA is a recently discovered negative modula-tor of the immune system [76]. VISTA is primarily expressed on hematopoietic cells, including APCs and T cells [77]. It is a suppressor of CD4 and CD8 T cells. In addition, within the CD4 subset, both effector and memory T-cells are effectively suppressed. T cells cultured with soluble VISTA-Ig fusion protein show no shift in expression of CD45RA to CD45RO [77]. Another notable phenomenon is that, while it blocks proliferation, it does not induce apoptosis and thus the cells remain viable. Lastly, the suppressive effect on T cells appears to be long lasting, even after VISTA effect was removed. In addition to being immunosup-pressive of T cells, it is also immuneregulatory. Under neutral conditions, VISTA-Ig is capable of suppressing naïve T-cells from forming Treg. This however was not the case when the culture conditions were changed, and in the presence of IL-2 and anti-CD28 VISTA-Ig was actually able to increase the proportion of Treg. Of note, VISTA does not appear to have any effect in vitro on B-cell proliferation or regulation. Its effect on cytokine production included reduced levels of IL-10, IFN-γ and TNF-α [77]. Anti-VISTA mAb exac-erbate experimental auto-immune encephalomyelitis

as well as enhancing antitumor immune responses [76]. Anti-VISTA mAbs are able to increase the number of tumor specific T cells in the periphery and enhance the infiltration, proliferation and effector function of tumor infiltrating lymphocytes within the tumor microenvironment [76]. In a melanoma model, both transplantable and inducible cancers are suppressed effectively with anti-VISTA monotherapy [78]. VISTA’s antitumor effect was also explored in concert with a peptide-based cancer vaccine where VISTA blockade synergistically impaired tumor growth.

CD27CD27 is a T-cell differentiation antigen and mem-ber of the TNFR superfamily [79]. It has increased membrane expression on anti-CD3-activated T cells. Agonistic CD27 mAb resulted in enhanced prolifera-tion of CD3 stimulated T cells. CD27 and its ligand CD70 are thought to have important effects on T-cell function [79]. Using intranasal influenza virus infec-tion as a model system, CD27 has been shown to be a major determinant of CD8 T-cell priming at the site of infection. CD27 signaling, along with other signal-ing including CD28, is crucial for the generation of antigen specific CD8 T cells. Via cell survival stimu-lation, CD27 promotes accumulation of activated T cells thereby expanding the proportion of virus spe-cific T cells [79]. The survival signal relies on IL-2R signaling and autocrine IL-2 production and CD27 is responsible for long-term survival of primed CD8 T cells, and hence memory. CD27 function has been extensively studied in mice and is transiently expressed during the germinal center reaction. CD27 expres-sion is most abundant during the phase of expansion of primed B cells and is absent from memory B cells. It appears that CD27 T cells provide help to B cells to form small germinal centers. Additionally, CD27 sig-naling in B cells results in enhanced levels of plasma cell formation and increased IgG production. Interest-ingly, constitutive CD27 signaling could have alternate effects on cellular and humoral immunity [79]. Collec-tively, the data elucidated CD27 signaling as a deter-minant of germinal center kinetics. In mouse models, costimulatory effect of CD27 was necessary for anti-CD40 antitumor efficacy [80]. Antitumor efficacy was shown in a mouse model of lymphoma using an anti-CD27 mAb. Anti-CD27 demonstrated no effect in SCID mice suggesting the need for an intact adaptive immune response and that the response itself was not due to a direct effect on the lymphoma cells [80].

A fully human anti-CD27 mAb, IF5, increased sur-vival in a mouse model of leukemia and lymphoma [81]. Its toxicity was assessed in a non-human primate model and has entered clinical development under the name



CDX–1127 (Varlilumab) and was assessed in a Phase I clinical trial [82]. The drug was well tolerated and responses included a complete response in a patient with stage IV Hodgkin’s lymphoma who had previ-ously failed stem cell transplant, chemotherapy and brentuximab-vedotin and three additional patients with stable disease.

OX40/OX40LOX40 is a member of the TNFR superfamily and is expressed on activated CD4 and CD8 T cells as well as other lymphoid and nonlymphoid cells [83]. It is also expressed in natural killer (NK) cells, NKT cells and neutrophils. OX40L is also a member of the TNFR superfamily and its expression is inducible on APC. Nonlymphoid cells can also be induced to express OX40L which supports of the role of the OX40/OX40L pathway in regulating the T-cell response. T cells themselves can express OX40L which repre-sents an additional mechanism for T-cell response amplification. TCR signaling is sufficient to induce OX40 in activated CD4 and CD8 T cells, however this is augmented by CD28 and B7-1/B7- 2 interac-tion and modulated by cytokines such as IL-1, IL-2 and TNF [83]. OX40L expression on the other hand is can be induced on APC upon activation by ligation of CD40 or by Toll-like receptors [83].

The OX40/OX40L pathway plays a large role in T cell expansion and survival, primarily by maintain-ing later proliferation and T-cell survival through the effector phase [83]. OX40 ligation can also directly inhibit naturally occurring Treg activity in mice pro-viding another means to promoting effector T-cell proliferation and survival [83]. Foxp3 expression on naïve CD4 T cells is blocked by OX40/OX40L activ-ity which supports a role in the suppression of naïve CD4 T-cells differentiation to become Treg. There have been conflicting reports however on the impact of OX40 signaling, which is expressed constitutively expressed on Treg, and may in fact promote Treg responses depending on the cytokine milieu [84].

The rationale for targeting OX40/OX40L signaling for cancer immunotherapy is supported by the expres-sion of OX40 in tumor infiltrating lymphocytes. Pre-clinical models have shown that injection of agonist OX40L-Ig fusion proteins, OX40 mAb, RNA aptam-ers that bind OX40 and transfection of tumor cells or DCs with OX40L can all suppress tumor growth [83]. In mouse models of cancer including sarcoma, mela-noma and glioma, among others, OX40 activity has been shown to decrease tumor growth [84]. The mecha-nism of tumor growth suppression is related to CD8 T-cell survival, and/or promotion of CD4 T-cell help for CD8 T cells. There may be an additional role via

augmentation of NK cell activity. Tumor infiltrating Tregs express high levels of OX40, and the signaling of OX40 within this environment suppressed their activ-ity [85]. In animal models, stimulation of OX40 appears to both augment antitumor activity as well as suppress Treg activity. Human studies include a Phase I trial using anti-OX40 mab 9B12 in patients with advanced cancers (NCT01644968 [86]) [87]. Therapy was well tol-erated with no maximum tolerated dose reached. Most common grade 3 or 4 side effects included lympho-penia that was transient. Best response to therapy by response evaluation criteria in solid tumors (RECIST) only included stable disease with some tumor regres-sion noted but less than 30% of overall tumor. One limitation of this agent was the induction of human antimouse antibodies, which precluded patients from receiving additional cycles. Nevertheless, this study provides evidence in humans that OX40 agonism can augment the immune system by stimulating CD4 and CD8 T-cell proliferation, CD8 IFN-γ production and increased antibody titers and T cell recall in response to tetanus immunization.

It seems that targeting OX40 alone may not be sufficient to elicit a robust antitumor response, and thus combination immunotherapy, particularly with antagonistic anti-CTLA-4 and anti-PD-1 antibodies, has been an area of study in preclinical models [84]. Combination therapies may stimulate complimen-tary pathways that synergistically respond to poorly immunogenic or large tumors, which has been an area of weakness in immunotherapy. Naturally, synergism may also extend to worsening the toxicity profile of such therapy but the fact the anti-OX40 therapy was fairly well tolerated may be promising.

CD40/CD40LCD40 and its ligand CD40L are members of the TNFR/TNF family [88]. CD40 is expressed on pro-fessional APC as well as other non-immune cells and tumors. Its ligand, CD40L is transiently expressed on T cells and other non-immune cells under inflammatory conditions [88]. Inherited lack of CD40L is responsible for x-linked hyper-IgM syndrome (H-XIM). It acti-vates DCs and allows them to stimulate CD8 T-cell activation and proliferation [89].

Binding of CD40L to CD40 promotes CD40 clus-tering on the cell surface, as well as recruitment of adapter proteins known as TRAFs to the cytoplasmic domain of CD40 [88]. CD40 signaling is carried for-ward by different pathways which include MAPKs, NF kappa B, PLC and PI3K. Additionally, it is noted that JAK3 binds the cytoplasmic domain of CD40 and can mediate other cellular processes. CD40/CD40L can directly activate DCs [89]. This CD40/CD40L inter-



action is necessary for the maturation and survival of DCs and CD40-dependent maturation of DCs leads to sustained expansion and differentiation of antigen specific T cells. The increased life span of DCs is very important in driving cell-mediated immunity. With-out the CD40 survival signal, passive apoptosis of T cell is induced [88].

CD40 signaling by B cells is required for the gen-eration of high titers of isotype switched high affin-ity antibody as well as for the development of humoral immune memory [88]. The binding of CD40L on CD4 T cells to CD40 on activated B cells is an important step in initiation and progression of the humoral immune response. Once CD40 signaling is active, there are downstream effects including B-cell intra-cellular adhesion, sustained proliferation, differentia-tion and antibody isotype switching. This process is essential for memory B cells and long lived plasma cells. B-cell fate is heavily influenced by CD40 sig-naling [88]. This signaling via binding of CD40 and CD40L can help determine whether the maturing B cell becomes a plasmablast or seeds a germinal center. With the activated CD40 pathway, the B cell will go on to form a germinal center. The lack of CD40 sig-naling is sufficient to block germinal center formation. T helper cells are recruited to these germinal centers, some of which express CD40L on their cell surface, which serve to maintain the germinal center. The lack of CD40 signaling in germinal center B cells increase Fas dependent apoptosis.

CD40 is expressed in mouse and human models of melanoma, prostate and lung cancers, and carcino-mas of the nasopharynx, bladder cervix and ovary [88]. Hematologic malignancies such as non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, acute leukemias and multiple myeloma also express CD40. Anti-CD40L mAb treatment inhibits the generation of protective immune responses from potent tumor vaccines [90]. Additionally, using CD40 deficient mice, no protec-tive antitumor immune response was induced follow-ing a protective vaccination regime. Early studies using a lymphoma model showed that agonistic anti-CD40 antibodies were able to eradicate tumor. Gene delivery of CD40L to DCs and tumor cells was sufficient to stimulate a long lasting systemic antitumor immune response in a murine model [90]. The approach of gene therapy with adenovirus expressing CD40L has been shown to be successful in colorectal carcinoma, lung carcinoma and melanoma murine models. CD40 ago-nism alone, however, was not sufficient for antitumor response likely due to the lack of TLR signaling.

Clinically, recombinant CD40L has been used in patients with solid tumors or non-Hodgkin’s lym-phoma given subcutaneously daily for 5 days in a

Phase I clinical trial [91]. Responses to therapy included 6% of patients with a partial response and one patient with a complete response. Humanized agonistic CD40 mAb include CP–870,893, SGN-40 and HCD 122. In a Phase I trial, CP-870,893 produced a par-tial response in 14% of patients (27% of melanoma patients). Dose-limiting toxicities were observed and included cytokine release syndrome [88]. Dacetuzumab (SGN-40) is a weak agonist and was studied in a Phase I trial in patients with refractory or recurrent B-cell non-Hodgkin’s lymphoma [92]. Toxicity was shown to be acceptable and antitumor activity was seen with six objective responses, 13 patients with stable disease. The overall response rate for patients in this cohort was 18%. In a Phase II trial with this drug, 46 patients again with relapsed or refractory diffuse large B-cell lymphoma were treated and the overall response rate was 9% and disease control rate (stable disease or better) was 37% [92]. Lucatumumab (HCD122) is a fully human anti-CD40 antagonist and was tested in 28 patients with refractory or relapsed MM [93]. Responses included 12 patients with stable disease, and one patient maintaining a partial response for greater than 8 months. It was also well tolerated with a good safety profile.

CD137(4-1BB)/CD137LCD137, also known as 4-1BB, is an induced T-cell costimulator molecule and a member of TNFR super-family [94]. CD137 is induced on activated CD4 and CD8 T cells, NK cells and constitutively on DCs, Tregs, monocytes and myeloid cells [94,95]. Its ligand 4-1BBL is expressed on B cells, DCs, macrophages, activated T cells and endothelial cells [94–96]. Ago-nistic CD137 mAb stimulated the proliferation of CD4 and CD8 T cells, mainly CD8 CTL, increased cytokine production, prevent activation induced cell death [94,97] and in absence of cognate signals increases the memory T-cell expansion [98]. Large tumors in mice are eradicated with increased cytotoxic T-cell activity in poorly immunogenic Ag104A sarcoma and highly tumorigenic P815 mastocytoma using agonist CD137 mAb [99]. CD 137 agonist mAbs also increase adhesion molecules ICAM-1, VCAM 1 and E-selectin thus increasing the trafficking of activated T-cells into tumor and also prevent immune tolerance by prevent-ing induction of CD8 CTL anergy to soluble tumor antigens [100].

Urelumab (BMS 663513) is humanized IgG4 mAb and PF 05082566 is humanized IgG2 mAbs for CD137 which are currently in clinical develop-ment [101,102]. BMS 663513 was tested in a Phase I/II trial with locally advanced and metastatic solid tumors. Initially patients with melanoma have been



enrolled but enrollment has been expanded to include renal cell and ovarian cancer patients [103]. Treatment is well tolerated and responses included three partial responses and four with stable disease [104]. A ran-domized Phase II trial (NCT00612664) in metastatic melanoma patients as a second-line therapy was termi-nated due to grade 4 hepatitis [95]. Urelumab is being tested with rituximab in B-cell non-Hodgkin’s lym-phoma or chronic lymphocytic leukemia as enhanced antibody-dependent cytotoxicity by rituximab was noted after activation of NK cells with CD137 [105]. Addition of anti-CD137 mAb to cetuximab improves efficiency of cetuximab in head and neck tumors as well as KRAS mutant and wild-type colorectal cancer, which provides further evidence for the use of immu-notherapy, specifically anti-CD137, in combination with other agents [106]. PF–05082566 was tested in a Phase I study of 27 patients and is well tolerated with mostly grade 1 adverse effects with one grade 3 elevated alkaline phosphatase was noted [107].

GITRGITR is a member of TNFR superfamily and is a co-stimulatory receptor. It has been originally discov-ered as up regulated in dexamethasone treated murine T-cell hybridomas [108]. It has very low expression in human T cells but constitutively expressed in human Treg [109]. Upon stimulation, naïve T cells and Treg upregulate GITR in a similar fashion to 4-1BB and OX40 suggesting their role in latter time points rather than early priming [110]. GITR-L is expressed in DCs, macrophages and B cells and is upregulated upon activation. GITR-L is also found in endothelial and activated T cells and may have role in leukocyte adhe-sion [110]. Its function is similar to OX40 and 4-1BB; it sends costimulatory signals inducing T-cell prolifera-tion, effector function and protects T cells from acti-vation induced cell death [110]. Combined anti-PD-1 blockage and GITR costimulation has potent anti-tumor activity in murine ID8 ovarian cancer model and is synergistic with chemotherapeutic agents. This combination promotes accumulation of CD4, CD8 T cells with decreased Treg and myeloid derived suppres-sive cells [111]. TRX518, an anti-GITR mAb, is being studied in a Phase I study in patients with stage III or stage IV melanoma or other solid tumor malignan-cies (NCT01239134 [86]). To circumvent autoimmune complications from immunomodulators mRNA trans-fected DCs are used locally to deliver anti CTLA-4 mAb and soluble GITR-L while increasing the anti-tumor immune responses [112]. Phase I clinical trial of a DC vaccine that entails intranodal injection of DCs transfected with mRNA encoding tumor antigens along with DCs transfected with mRNA encoding

soluble human GIRT-L and anti CTLA-4 mAb is in progress (NCT 01216436 [86]).

Tim-3Tim-3 was first described in 2002 [113]. It is expressed on CD4 T cells and CD8 CTLs. Tim-3 binds several molecules including Gal-9, CEACAM1, HMGB1 as well as glycosylated molecules [114]. Binding of its ligand, Gal-9 induces cell death and thus illustrating its role as a negative regulatory molecule, with particular importance in Th1- and Tc1-driven responses. Tim-3 is expressed on all IFN-γ secreting Th1 cells as well as DCs [113]. Th1 immunity is regulated via binding of its ligand, Gal-9, directly triggering cell death. It has also been shown that Tim-3/Gal-9 binding also suppresses immune responses indirectly by expanding the popula-tion of myeloid derived suppressor cells [113]. Tim-3 has been implicated in inducing T-cell exhaustion in sev-eral scenarios including chronic viral infections such as hepatitis C and HIV, bacterial infections and can-cer [113]. In murine models of colon adenocarcinoma, melanoma and mammary adenocarcinoma, Tim-3 can be co-expressed with PD-1 in tumor infiltrating CD4 and CD8 T cells. Tim-3+PD-1+ CD8 T cells were among the most impaired T cells with reduced prolif-eration and decreased production of IL-2, TNF and IFN-γ. In vivo, Tim-3 blockade in concert with PD-1 blockade produced a significantly higher antitumor effect than either one alone. Additionally, this com-bined blockade increased the frequency of proliferating antigen specific CD8 T cells.

LAG-3LAG-3, also called CD223, is expressed on acti-vated T cells, NK cells, B cells and tumor infiltrat-ing lymphocytes [115]. It is closely related to CD4; it is a member of the immunoglobulin superfamily and its gene is located near CD4 on chromosome 12. LAG-3 is a negative regulator of T-cell activation and homeostasis [115]. LAG-3 binds to MHC class II mol-ecules with high affinity [116]. LAG-3 cross-linking on activated human T cells induces T-cell functional unresponsiveness and inhibits TCR-induced calcium ion fluxes. Similar to CD4 and CD8, LAG-3 is con-sidered to be a coreceptor to the CD3–TCR complex. Inhibition of cytokines, such as IL-2, is induced by LAG-3 in vitro. It decreases the pool of memory CD4 and CD8 T cells. It also increases the suppres-sive activity of Treg. For maximal suppressive activ-ity of Treg, LAG-3 signaling is required [117]. It was not clear however, if the signal alone was sufficient. Within the tumor microenvironment LAG-3 pro-motes immune tolerance of the tumor by inhibiting APC and T-cell function [118].



Malignant mouse and human tissue has been shown to co-express PD-L1 and LAG-3 [119]. A sig-nificant percentage of tumor infiltrating CD4 and CD8 T cells from mouse tumor models of melanoma, colorectal adenocarcinoma and fibrosarcoma, express high levels of LAG-3 and PD-1. When using anti-LAG-3 immunotherapy, reduced growth of fibrosar-coma and colorectal adenocarcinoma is observed in some mice. This same effect is seen with anti-PD-1 monotherapy. Anti-LAG-3 produced a synergistic effect when combined with anti-PD-1 immuno-therapy with 70% of the fibrosarcoma and 80% of colorectal adenocarcinoma mice noted to be tumor free [119]. This regimen, however, was shown to have no effect on the melanoma model. Interestingly, treating mice with anti-LAG-3/anti-PD-1 combined therapy is proposed to be less toxic given that LAG-3 and PD-1 co-expression is largely limited to tumor infiltrating lymphocytes.

IMP321 is a clinical grade LAG-3-Ig recombinant fusion protein that antagonizes normal LAG-3 func-tioning [120]. In 2009 the results of the first Phase I study involving IMP321 alone was released. Patients with advanced renal cell carcinoma were treated [121]. No significant adverse events occurred. Tumor growth reduction was seen and progression-free survival was better in those patients receiving higher doses (>6 mg). Out of eight patients treated with high dose IMP321, seven had stable disease at 3 months com-pared with only three of 11 in the lower dose group. Another Phase I trial of IMP321 and paclitaxel in metastatic breast cancer was conducted [122]. Patients treated with IMP321 were found to have a sustained increase in the number and activation of monocytes and DCs as well as an increase in the percentage of NK and long lived cytotoxic effector memory CD8 T cells, which correlates well with the preclinical data. Additionally 90% of patients had some clini-cal benefit, with only three of 12 patients progress-ing by 6 months. Objective tumor response rate was 50% compared with the historic control group of 25% [122]. IMP321 is well tolerated as well. There were several other Phase I trials combining IMP321 with other agents. In 18 patients with advanced pan-creatic adenocarcinoma, IMP321 was combined with conventional gemcitabine and had a good safety pro-file [123]. Its clinical benefit was hard to evaluate likely due to suboptimal dosing of gemcitabine. IMP321 was combined with an anticancer vaccine MART-1 peptide and used in 12 patients with advanced mela-noma [124]. Six patients received MART-1 alone and six in combination with IMP321. One patient expe-rienced a partial response in the IMP321 group and none in the MART-1 alone group.

BTLABTLA (CD272) is a transmembrane protein that is expressed on Th1 cells as well as B cells and DCs [125–127]. BTLA, via interaction with HVEM (her-pes virus entry mediator), inhibits cancer specific CD8 T cells [127]. HVEM is expressed in hematopoietic cells, including B and T cells, as well as in nonhematopoietic cells (parenchymal cells) [126]. HVEM is also expressed in melanoma cells and variety of solid tumors [126]. Hodgkin’s lymphoma, B-cell non-Hodgkin’s lym-phoma and some T-cell non-Hodgkin’s lymphomas use BTLA for immune evasion [127]. BTLA and HVEM are highly expressed in B-cell chronic lymphocytic leukemia suggesting their role in pathogenesis [126,128]. BTLA–HVEM is implicated in Vγ9Vδ2 T-cell prolif-eration, differentiation and has a critical role in their control of lymphogenesis [129]. T-cell responses against minor histocompatibility antigens on malignant cells play an important role for cure in hematological malig-nancies after allogeneic stem cell transplant via graft versus tumor effect. BTLA suppresses minor histocom-patibility antigen-specific CD8 T cells after allogeneic stem cell transplant thus providing a rationale for its clinical utility in post transplantation therapies [130]. High BTLA expression is associated with shorter sur-vival in gastric cancer [131]. BTLA expression is upregu-lated on cytotoxic CD8 T cells in peripheral blood of patients with hepatocellular carcinoma and correlates with disease progression and increased expression is associated with high recurrence rates [132]. Downregu-lation of BTLA in vivo can be attained by adding CpG oligonucleotides to vaccine formulation, which leads to increased resistance to BTLA/HVEM inhibition [133]. Because of the role of BTLA/HVEM in hematological as well as solid tumors, BTLA inhibition also represents a target for cancer immunotherapy.

IDO synthaseTryptophan plays an important role in peripheral immune tolerance through its rate limiting tryptophan degradation along the kynurenine pathway, including IDO1 and tryptophan-2,3-dioxygenase (TDO). Short-age of tryptophan leads to cell cycle arrest, decreased proliferation through inactivation of mTOR pathway, while tryptophan metabolites can cause T-cell apop-tosis, and induce differentiation of Tregs. Tumors can evade the immune system by hijacking tryptophan catabolizing enzymes IDO1 and TDO [134]. IDO1 protein is expressed in mature DCs in lymphoid tis-sues, some epithelial cells of female genital tract, pla-cental and pulmonary endothelial cells [135]. IDO1 positive cells are scattered in human and tumoral lym-phoid tissues particularly in cervical, colorectal and gastric carcinomas. It is highly present in vascular cells


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in renal cell cancer [135]. Expression of IDO1 is highest in endometrial and cervical cancers followed by kid-ney and lung [135]. IDO1 expression is associated with aggressive phenotype, poor prognosis, shorter survival and increased Tregs [136]. IDO1 inhibitors were shown to exhibit antitumor effect in mouse models alone as well as have synergistic effects with a variety of che-motherapeutic agents in preclinical models [137–140]. Indoximod is an IDO inhibitor that is being studied in a Phase II trial with taxane chemotherapy in meta-static breast cancer (NCT 01792050 [86]). IDO inhibi-tors can provide a synergistic effect when administered with vaccines and immunotherapy; IDO induction in response to inflammation can attenuate antitumor vaccine [139]. This provided the rationale for a random-ized Phase II study of Indoximod with Sipuleucel-T (Provenge®) in the treatment of patients with asymp-tomatic or minimally symptomatic metastatic castra-tion resistant prostate cancer (NCT01560923 [86]). Other studies include a Phase I study of NLG–919, an IDO inhibitor, for patients with advanced solid tumor malignancies and a Phase I/II trial of the indoximod in combination with ipilimumab for the treatment of unresectable advanced stage melanoma [141,142].

Conclusion & future perspectiveOur understanding of immune dysfunction in cancer continues to develop. Growth and progression of can-cer are made possible by the ability of the malignant cells to manipulate immune checkpoint pathways that prevent immune overstimulation. Significant progress has been made in the field with mAb against CTLA-4 and now PD-1 in clinical use. This gives credence to the efforts aimed at developing agents targeting other immune modulatory pathways. The success PD-1 inhibitors may very well translate to PD-L1 inhibi-tors being successful in the clinic. PD-L1 is widely expressed in a variety of tumors and PD-L1 inhibitors have shown impressive, albeit preliminary, results in areas of unmet need such as urothelial bladder can-cer which has been resistant to conventional chemo-therapy [34,50]. Other B7 family members are emerg-ing as clinical target in preclinical models such as B7x, HHLA2 and B7-H3. Other immunoglobulin super-family members such as Tim-3 and VISTA also show promise in preclinical models. LAG-3 targeting mol-ecules have also reached clinical trials with good safety profile and evidence of antitumor efficacy.

TNFR superfamily member also hold promise in cancer immunotherapy. Preclinical and clinical data support targeting CD27 where a durable response was noted as well [81,82]. mAb targeting CD40L have reached clinical trials although the clinical experience in the CD40/CD40L pathway illustrates some of the

caution needed with immune disinhibition, particu-larly in unwanted side effects such as cytokine release syndrome [88,92,93]. OX40, CD137 and GITR antibod-ies have also reached clinical trials providing further evidence that TNFR family members are viable targets for cancer immunotherapy [101–104,112]. Table 1 is the summary of clinical trials with agents targeting immu-nomodulatory pathways beyond CTLA-4 and PD-1/PD-L1.

The redundancy in inhibitory immune checkpoint molecules sheds light on the complexity of immune regulation. It also allows for the targeting of these mol-ecules in succession or in combination. A new path-way may be targeted after the efficacy of an earlier immunotherapy is exhausted as evidenced by the suc-cess of PD-1 targeting mAbs in ipilimumab-refractory tumors indicating that tumors may recruit additional pathways when one is blocked, or several pathways may be in fact be recruited simultaneously by tumor cells [24,25,31]. Efficacy of ipilimumab in combina-tion with nivolumab also illustrates that tumors may recruit more than one inhibitory pathway at the same time [143].

Another interesting dilemma that emerges as cancer immunotherapy gains momentum is integrating these novel agents with current regimens that mainly consist of conventional cytotoxic chemotherapy. On one hand, conventional wisdom may lead us to believe that che-motherapy may be synergistic. The immunosuppressive effect of chemotherapy however can have unpredict-able effects on the immune system. Cyclophosphamide has been used as a Treg depleting agents in preclini-cal models with some success [144,145]. Combination regimens have also reached clinical trials [34,122,123]. Combination of immune checkpoint inhibitors have also been studied with traditional immunotherapies or targeted therapies such as rituximab and cetuximab where they show efficacy and tolerability [105,106,146]. With evidence pointing to better outcomes in second line setting or even first line setting of immunothera-pies, cancer immunotherapy will play a vital role in the future of oncology [24,31,147].

Financial & competing interests disclosureResearch in the Zang lab is supported by NIH R01CA175495,

Department of Defense Established Investigator Idea De-

velopment Award PC131008, and Dr Louis Sklarow Memo-

rial Trust. The authors have no other relevant affiliations or

financial involvement with any organization or entity with a

financial interest in or financial conflict with the subject mat-

ter or materials discussed in the manuscript apart from those

disclosed.


manuscript.



ReferencePapers of special note have been highlighted as: • of interest


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Executive summary

Cancer immunotherapy targets in clinical trials• PD L-1 blockade can restore cytotoxic T-lymphocyte antitumor activity.• CD27 is a hematologic malignancy marker that has been targeted in Phase I trials.• Lag-3 is a co-inhibitory molecule and blocking antibodies have reached clinical trials.• CD40, CD137 and GITR agonism may play a role in cancer immunotherapy and have reached Phase I and

Phase II trials.• Indoleamine 2,3-dioxygenase is not a cell-surface protein and inhibitors have reached clinical trials.Emerging targets for cancer immunotherapy still in preclinical study• B7x, HHLA 2, B7-H3 are B7 family members that have inhibitory role and are expressed by tumor cells.• Tim-3, VISTA and BTLA blockade are emerging targets in preclinical models.• Agonism of stimulatory molecules OX40 and OX40L may also play a role in cancer immunotherapy.Conclusion• T-cell disinhibition is now a clinically effective approach.• Targeting of several pathways may be done in succession or in concert.• Immunotherapy can be more effective than chemotherapy especially in the second-line setting.



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REVIEW

Emerging immunotherapy in pediatric lymphoma

Craig Erker1, Paul Harker-Murray1 & Michael J Burke*,1

1Division of Pediatric Oncology, Medical College of Wisconsin, Milwaukee, WI 53226, USA

*Author for correspondence: Tel.: +1 414 955 4170; Fax: +1 414 955 6543; [email protected]

Hodgkin and non-Hodgkin lymphoma collectively are the third most common cancer diagnosed in children each year. For children who relapse or have refractory disease, outcomes remain poor. Immunotherapy has recently emerged as a novel approach to treat hematologic malignancies. The field has been rapidly expanding over the past few years broadening its armamentarium which now includes monoclonal antibodies, antibody–drug conjugates and cellular therapies including bispecific T-cell engagers and chimeric antigen receptor-engineered T cells. Many of these agents are in their infancy stages and only beginning to make their mark on lymphoma treatment while others have begun to show promising efficacy in relapsed disease. In this review, the authors provide an overview of current and emerging immunotherapies in the field of pediatric lymphoma.

First draft submitted: 18 August 2015; Accepted for publication: 13 October 2015; Published online: 30 November 2015

KEYWORDS • checkpoint inhibitors • Hodgkin lymphoma • immunotherapy • non-Hodgkin lymphoma • pediatric

Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) are the third most common pedi-atric cancers in the USA with around 2000 cases diagnosed each year [1,2]. Although 5-year overall survival (OS) in patients with HL exceeds 90%, delayed therapy-related toxicity reduces long-term survival [1]. Among the 10–20% of HL patients with relapsed or refractory disease [3], survival remains near 50% despite high-dose chemotherapy and autologous stem cell rescue (ASCR) [1]. Similar to HL, pediatric NHL has cure rates ranging from 70% to greater than 90% depending on stage and subtype. By contrast, the prognosis for children with relapsed NHL remains very poor with 5-year event-free survival (EFS) of approximately 30% with traditional chemotherapy [4]. High-dose chemotherapy followed by ASCR results in 5-year EFS of approximately 30–50% for patients with Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL) [5], and 5-year EFS is approximately 40% for patients with lymphoblastic lymphoma (LL) following allogeneic stem cell transplantation (allo-HCT) [5]. For those who survive, long-term toxicities remain a significant cause of morbidity and late mortality [6].

Immunotherapy has gained considerable interest for the treatment of both HL and NHL in children with the goal of improving outcomes and decreasing the long-term toxicities of treat-ment. Attempting to harness the immune system to fight cancer was first demonstrated in 1982 when Miller and colleagues reported an antitumor response with patient-derived monoclonal antibodies (mAbs) in an adult patient with a nodular, poorly differentiated lymphocytic NHL variant [7]. mAbs for pediatric lymphoma were reported shortly after in two children, ages 22 and 30 months, with post-transplant lymphoproliferative disease (PTLD), where anti-CD24 and anti-CD21 mAbs were successfully administered, achieving remission in both children within


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3 weeks of treatment [8]. Since then, mAbs tar-geting different tumor antigens have been devel-oped, modified and improved with the addition of conjugated immunotoxins and radiolabeled isotopes. Newer classes of antibodies, known as immune checkpoint inhibitors, have been devel-oped which can disinhibit tumor suppression of the immune response. In addition, recent novel cell-based therapies include the bispecific T-cell engagers and chimeric antigen receptor T-cell therapy which both harness the patient’s T cells to fight the cancer. In this article, we review cur-rent and emerging immunotherapies, along with the early-phase studies which introduced them into the lymphoma arena and identify those for which pediatric studies have been developed.

mAb therapy●● Monoclonal antibodies

mAbs directed against tumor-specific antigens were approved for treatment of lymphomas in adults prior to 2000. Antitumor activity may occur through a number of mechanisms includ-ing antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxic-ity (CDC) and by the induction of apoptosis through caspase activation and sensitization to chemotherapy of resistant lymphoma cells [9–11]. CDC is triggered when the Fc domain binds and activates the complement cascade, leading to formation of the membrane attack complex (MAC), which disrupts the tumor cell’s phos-pholipid bilayer and results in cell lysis and death. Immune effector cells such as mono-cytes, natural killer (NK) cells or granulocytes have Fc receptors on their surfaces which rec-ognize antibody coated tumor cells and patho-gens. The cross-liking of Fc receptors triggers cell destruction via localized degranulation (ADCC) or engulfment by phagocytosis [12]. The initial mAbs were human/murine hybrids. The immunogenicity of these chimeras resulted in infusion reactions, hypersensitivity reactions and the development of neutralizing antibodies in some patients. However, these complications have become less common as subsequent genera-tions of mAbs have been fully humanized [13]. Antitumor antibodies have been subsequently modified by conjugation with immunotoxins or by adding radiolabeled isotopes. In addition newer agents have included novel mechanisms of immune stimulation via checkpoint inhibition.

Mature B-cell lymphomas, including BL, DLBCL, HL and primary mediastinal B-cell

lymphoma (PMBCL) commonly express sur-face antigens such as CD20, CD22 and CD30 in high density making them excellent targets for antibody-directed therapy. mAbs directed against CD20 can be divided into two subtypes based on their CD20 epitope binding and mech-anism of action [14]. Type I mAbs induce redistri-bution of CD20 molecules into large rafts on the cell surface. This results in clustering of the Fc regions which enhances C1q recruitment, com-plement activation and subsequent CDC [15]. By contrast, type II mAbs do not induce raft formation and are therefore relatively ineffective in CDC. Instead, they induce direct cell killing by induction of apoptosis and indirect killing through ADCC [14].

Rituximab is a type I CD20-directed chimeric (murine/human) mAb that has been well estab-lished as therapy for mature B-cell lymphomas in adults. In multiple studies, rituximab has been shown to improve both event free and OS when given in conjunction with standard chemother-apy [16–19]. Similarly it has shown significant activity in adults with nodular lymphocyte pre-dominant HL (NLPHL) [20,21] In a small meta-analysis of 24 pediatric patients, median age 9.5 years (range: 3–20), with relapsed/refractory BL treated with rituximab alone or in combi-nation with intensive chemotherapy, 12 of 24 (50%) achieved a complete remission (CR) while 5/24 achieved a partial response (PR) [22]. In a pilot study by the Children’s Oncology Group (COG), rituximab has been shown to be safe and tolerable when added to conventional chemotherapy in pediatric patients with high-risk mature B-cell lymphomas with grade 3 mucositis reported in 31% and grade 4 in 26% of patients [23]. Although this study was not powered to show improved outcomes compared with historical controls, the EFS at 3-years was 90% (95% CI: 76–96). Currently there is an ongoing international trial to determine the efficacy of rituximab with chemotherapy in stage III–IV mature B-cell lymphoma or B-cell acute leukemia (NCT01595048) [24].

Ofatumumab is a type I humanized CD20-directed mAb which binds to a different epitope than rituximab. Compared with rituximab, ofa-tumumab results in greater complement activa-tion and antibody-dependent phagocytosis [14]. However, this has translated into only modest clinical activity when used as monotherapy in adults with relapsed or refractory DLBCL [25]. Currently there are two Phase II pediatric studies

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investigating ofatumumab in BL and B-cell LL (NCT02419469; NCT02199184) [26,27].

Obinutuzumab is a humanized, type II mAb whose Fc region has been glycoengineered to have an enhanced affinity for the FcγRIIIa pro-tein leading to more effective ADCC [28]. In an adult Phase I study of single agent obinu-tuzumab, 21 patients with relapsed/refractory NHL received obinutuzumab on days 1 and 8 for cycle one and on day 1 for each subsequent cycle [29]. In this trial, five patients reported a CR and four a PR for an overall response rate (ORR) of 43%. The most common reported adverse events (AEs) were infusion-related reactions with the first cycle. Currently obinutuzumab is being combined with ifosfamide, carbopla-tin and etoposide in pediatric and young adult patients with CD20-positive lymphoma in first relapse or induction failure (NCT02393157) [30].

Epratuzumab is a humanized mAb against CD22, an antigen that is present on >95% of all B cells [31]. Epratuzumab has been combined with chemotherapy in adults with DLBCL as well as in combination with rituximab plus chemotherapy (ER + CHOP) [32,33]. Toxicities of ER + CHOP were similar to what is reported with R-CHOP alone with febrile neutropenia (16%), vomiting (29%), fatigue (36%), hyperglycemia (17%) and cytopenias (93%) being most common. Because CD22 has the potential to be rapidly internal-ized upon ligation [34], epratuzumab has been radiolabeled with Yitrium90 and conjugated with the chelator tetraxetan to further enhance radio-isotope delivery to tumor cells. This strategy has shown promising activity in adults with relapsed/refractory NHL reporting response rates as high as 71% in patients without a prior ASCR and 41% in those who had previously failed ASCR [35]. Other than two cases of grade 4 cytopenia, the AEs were all grades 1 or 2, with the more com-mon toxicities being infection (31%), asthenia (17%), nausea (16%), cough (11%), fever (9%) and constipation (8%). Although there have been no pediatric lymphoma trials to date investigat-ing epratuzumab, the COG reported reasonable activity in patients with relapsed acute B-cell lymphoblastic leukemia (B-ALL) [36].

●● Conjugated immunotoxin mAbsAntibody–drug conjugates (ADC), also known as conjugated immunotoxins, are mAbs cova-lently linked to an anticancer agent (e.g., cali-cheamicin) designed to target tumor surface proteins, become endocytosed and deliver a

cellular poison directly to the cancer cell. These mAbs were developed to be more efficient than the traditional naked mAbs in cell killing and as a cancer immunotherapy [37].

Brentuximab vedotin (SGN-35) is an anti-CD30 antibody linked to monomethyl aurista-tin E (MMAE; vedotin) via a protease-cleavable linker. Upon release MMAE binds to tubulin and disrupts the microtubule network result-ing in cell cycle arrest and apoptosis [37]. In adults with relapsed/refractory CD30-positive ALCL, single agent brentuximab vedotin had an impressive 86% ORR and 57% CR rate [38]. The reported grade ≥3 toxicities occurring in greater than 10% of patients included neutro-penia (21%), thrombocytopenia (14%) and peripheral sensory neuropathy (12%). Although there have been no pure pediatric Phase I trials of brentuximab vedotin published to date, several early-phase studies in CD30-positive lympho-mas have included pediatric patients down to age 12–14 years [38–40]. Three pediatric trials are currently assessing the efficacy of brentuximab vedotin with combination chemotherapy for the treatment of newly diagnosed CD30-positive ALCL (Phase III), high-risk HL (Phase III) and relapsed HL (Phase I/II; NCT01979536, NCT01780662, NCT02166463) [41–43].

Inotuzumab ozogamicin is a humanized anti-CD22 ADC linked to the toxin calicheam-icin, an antitumor antibiotic which binds to the minor groove of DNA leading to double-stranded DNA breaks [44]. Inotuzumab has been combined with rituximab (R-INO) for adults with relapsed/refractory CD22-positive DLBCL and reported response rates of 28.6% after three cycles of R-INO. The most common toxicities seen with this combination included thrombocytopenia, lymphopenia and neutro-penia [45]. Although pediatric BL and DLBCL often express CD22, there are no current trials for this agent in pediatrics.

Polatuzumab vedotin is an ADC targeting CD79b which is expressed by the vast majority of B cells as well as B-cell NHL including both BL and DLBCL. Similar to brentuximab vedotin, polatuzumab vedotin is linked to the microtu-bule-disrupting agent MMAE. In a recent Phase I trial of single-agent polatuzumab vedotin, 45 adult patients with relapsed/refractory NHL were assigned to an expansion cohort at the recom-mended Phase II dose with objective responses reported in 23 of 42 (54.8%) evaluable patients and seven of the 23 (30.4%) achieving a CR [46].

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Twenty-five of the 45 patients in this study had a diagnosis of DLBCL where 14/25 (56%) had an objective response. Grade ≥3 toxicities occurred in 58% of patients (n = 26) with the most com-mon events being neutropenia (40%), anemia (11%) and peripheral sensory neuropathy (9%). As with inotuzumab ozogamicin, there are no current pediatric l ymphoma trials investigating p olatuzumab vedotin.

Moxetumomab pasudotox is an ADC where the Fv portion of an anti-CD22 antibody is fused to PE38, a recombinant immunotoxin derived from the pseudomonas exotoxin A, which induces apoptosis by inhibiting protein synthe-sis [34]. In a Phase I study of 21 pediatric patients with relapsed/refractory B-ALL treated with sin-gle agent moxetumomab pasudotox, 17 patients were evaluable for response with four (24%) achieving a CR and one (6%) a PR [47]. The most common AEs were increased weight, elevated ALT/AST and hypoalbuminemia. Currently for pediatric lymphoma, there has been one recently completed trial in relapsed/refractory NHL (NCT00659425) and another investigating moxetumomab pasudotox in relapsed/refractory B-ALL or B-LL (NCT02227108) [48,49].

●● Bispecific T-cell engager antibodiesBispecific T-cell engager (BiTE®) antibodies use a technique that capitalizes on a T cell’s cyto-toxicity with direct cell–cell killing. These anti-bodies are engineered to have two variable anti-body domains; one binding the tumor antigen (e.g., CD19) and the other binding an activating antigen on the cytotoxic effector cell (e.g., CD3) (Figure 1) [14]. These antibodies induce an immu-nologic synapse between an endogenous cyto-toxic T cell and the cancer cell [50] resulting in T-cell degranulation and release of perforin and granzyme. Unlike a typical cellular immune response in which less than one in 105 to 106 T cells have T-cell receptors with specificity for tumor antigens, bispecific T-cell engager anti-bodies trigger T-cell activation, which is inde-pendent of T-cell receptor antigen specificity [51]. Blinatumomab is the model example of BiTE technology. Blinatumomab cross-links CD19 on tumor cells and CD3 of the patient’s cyto-toxic T cells and has shown promising activity in both pediatric and adult B-ALL reporting CR rates over 60 with 88% of responders achiev-ing minimal residual disease negativity [51]. Blinatumomab is also active against follicular and mantle cell lymphoma in adults and its use has

expanded to studies in adult relapsed/refractory DLBCL [52]. In a study of 11 evaluable patients with DLBCL receiving blinatumomab, six (55%) patients showed a response of which four achieved a CR [53]. The most common AEs were fever (82%), fatigue (55%), constipation (36%), headache (36%), tremor (36%) and weight gain (36%). Currently, blinatumomab has not been tested in pediatric lymphomas but the promising results in pediatric ALL and adult NHL make it an encouraging agent to bring forward into pediatric B-cell lymphoma therapy.

Other BiTE antibodies that are in develop-ment include the humanized CD20 T-cell-dependent bispecific antibody (CD20-TDB) which is highly active against primary B-ALL and lymphoma cells in both in vitro and in vivo studies [54]. Another bispecific antibody is AFM13 (CD30/CD16A) which binds to the CD30 antigen, which is expressed by ALCL and the Reed-Sternberg cells of HL, and to the CD16A antigen, an activating receptor on NK cells [55]. In a trial of 26 evaluable adult patients with relapsed or refractory HL, AFM13 had an ORR of 11.5% with 3 (10.7%) patients achiev-ing a PR and 13 (50%) achieving stable disease (SD) [55]. The treatment was very well tolerated with most AEs being mild to moderate. The most common events reported in at least four patients included fever (54%), chills (39%), headache (29%), nausea (18%), infusion reac-tion (14%), rash (14%), vomiting (14%) and pneumonia (14%).

●● Radiolabeled mAbsRadioimmunotherapy (RIT) involves mAbs that deliver therapeutic doses of radionuclides to a targeted site such as a tumor cell. Over the past 30 years, considerable improvements have been made in the development of RIT including use of recombinant and humanized mAbs, synthesis of more stable chelates for radiolabeling and the improvement of pretargeting techniques [56,57]. RIT combines the cytotoxic effects of mAbs in inducing apoptosis with additional radiobiologi-cal cytotoxicity mechanisms via their conjugated radioisotope [56].

Iodine 131 (131I) tositumomab is an example of RIT composed of a murine anti-CD20 antibody linked to the radioisotope 131I which emits both β and γ radiation. This technology utilizes the spec-ificity of the CD20 mAb targeting B-cell lympho-mas with the underlying radiosensitivity of NHL to achieve a combined antitumor effect [58]. No

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Figure 1. BiTE® antibodies mediate the contact between the host T cells and tumor cells while engineered CAR T cells target tumor cells with the desired antigen. In both cases, these mechanisms harness cytotoxic T cells via activation and release of perforin and granzyme leading to tumor cell death (i.e., apoptosis).

Target antigen(i.e., CD19)

Tumor cell

Perforin Apoptotic body

BiTEscFv

scFvscFv(anti-CD3-IgG)

CD3deCD3z

CytotoxicT cell

Granzymes

CAR T-cellsecond generation

4-1BBCostimulationdomain



pediatric studies currently exist for tositumomab however several have been completed in adult HL and NHL. One adult Phase III study of 224 patients with relapsed/refractory DLBCL com-pared withsitumomab plus BEAM (carmustine, etoposide, cytarabine, melphalan) chemotherapy to rituximab and BEAM prior to ASCR [59]. Although this study showed the feasibility of this approach, there was no survival advantage at 2 years between groups. The primary toxicity compared between treatment arms was mucosi-tis which was found to be greater in the tositu-momab arm. Tositumomab was removed from the market in 2013 due to declining sales and the availability of other RIT agents [60].

Another 131I-labeled RIT agent (Ki-4), a murine anti-CD30 mAb, was assessed in 22 adolescent and young adult patients with relapsed/refractory HL (median age of 31 years; range: 15–43) [61]. Patients received total doses of radiation ranging from 0.035 to 0.99 Gy. Treatment responses included one CR and five PRs. Although the overall acute toxicities from Ki-4 were minimal, seven patients reported severe grade 4 hematologic toxicity 4–8 weeks after treatment and this has limited its ability to proceed further in clinical trials.

Yttrium-90/90Y-ibritumomab tiuxetan (90Y-IT) is another anti-CD20 murine anti-body linked to a radionuclide. The antibody is covalently joined to the chelating agent tiuxetan (MX-DTPA) which in turn tightly binds radio-nuclides. 90Y is a pure β-emitting isotope, five-times more energetic than 131I, and delivers 90% of its energy within 5 mm of disintegration [62,63]. The COG performed a Phase I study using 90Y-IT plus rituximab in children with relapsed/refractory CD20-positive NHL [62]. Five patients received rituximab and 90Y-ibritumomab with no patients experiencing a dose limiting toxic-ity (DLT). Reported toxicities included grade thrombocytopenia (n = 2), grade 4 neutropenia (n = 2), grade 3 anemia (n = 1) and grade 3 infec-tion (n = 1). The mean radiation exposure in patients ranged from 42 cGy (total body) to 565 cGy (spleen); however, no objective responses were reported.

131I-F16SIP (Tenarad) is a fully humanized RIT specific to the alternative spliced A1 domain of tenascin-C, an extracellular matrix molecule that modulates angiogenesis, cell signaling, migration, proliferation and metastasis [64]. The A1 domain of tenascin-C is largely undetectable in normal tissues, but present in many tumor

Future Oncol. (2016) 12(2)262



types making it suitable for radiotherapy [57]. This drug has been tested in HL where neo-plastic growth is highly regulated by interactions between the tumor cells and their surrounding angiogenic signals that promote tumor growth and survival [57]. In a study of eight young adult patients (ages 19–44 years) with progressive HL, monotherapy with 131I-F16SIP (Tenarad) resulted in one CR, one PR and five patients with stable disease [57]. Moderate-to-severe (grades 2 and 3) thrombocytopenia, neutropenia and lym-phopenia was reported in all patients. Although no patients were ultimately cured with this therapy, it appeared effective in chemorefrac-tory patients resulting in objective responses and was well tolerated. See Table 1 for a more c omprehensive list of mAbs, ADC and RIT.

●● Therapies using T cellsOne potential disadvantage of using mAbs as cancer immunotherapy is their relatively short half-life and therefore transient effect as the anti-body may be quickly metabolized and cleared from circulation. To overcome this potential therapeutic hurdle, cellular therapies have been engineered which redirect the host’s cytotoxic T cells against hematologic malignancies with impressive and sustained results. Adoptive cel-lular transfer (ACT) is an example of this and includes the ex vivo alteration and expansion of autologous or allogeneic T cells that target specific tumor surface molecules [54]. Both HL and NHL can express Epstein–Barr virus (EBV) antigens which serve as targets for adoptive T-cell therapy [69,70]. In a pediatric and young adult study, 14 patients (ages 8–40 years) with relapsed/refractory HL were treated with in vivo expanded EBV-specific cytotoxic T lymphocytes (CTLs) [71]. The infused CTLs were very well tolerated with two patients reporting transient flu-like symptoms, otherwise no AEs were attrib-uted to the CTL therapy. The CTLs persisted in the circulation for up to 12 months, and resulted in a CR in five patients (two maintaining a CR for up to 40 months), PR in one and SD in another five patients. There are currently sev-eral active pilot, Phase I and Phase II CTL stud-ies available for children and young adults with lymphoma (NCT02057445; NCT01555892; NCT02287311; NCT01956084; NCT01447056 and NCT01636388) [72–77]. As up to 70% of HL can be EBV negative, nonviral tumor-associated antigens have also been targeted for cellular therapy. Conrad et al. has shown that a subset

of EBV-negative HL expresses MAGE-A4 and this expression is enhanced by pretreating with the demethylating agent decitabine prior to infu-sion of MAGE-A4 specific T cells [70]. A Phase I study is currently underway in adults with HL and NHL to study the efficacy of CTLs directed against several tumor-associated antigens (TAAs) including MAGE-A4, NY-ESO-1, PRAME, Survivin and SSX (NCT01333046) [78].

Chimeric antigen receptor (CAR) T cells are genetically engineered CTLs using viral vec-tors, DNA transposons or RNA transfection to express a T-cell activating receptor for a tumor antigen. CARs consist of three domains: an extracellular antigen-binding domain, a trans-membrane domain and at least one intracellular signal transduction domain [79]. Thus when a CAR T cell binds to a tumor antigen, the intra-cellular signaling domain is activated leading to a tumor cell death process by the activated T cells (Figure 1). There are currently three differ-ent recognized generations of CAR T cells. The first generation of CAR T cells had a relatively poor effectiveness as they carried only a primary T-cell receptor signal activation domain con-sisting of either CD3-ζchain or FceRIγ [79,80]. Second generation CAR T-cells have an addi-tional costimulatory molecule, either CD28 or CD137 (4–1BB), which has improved T-cell activation [80]. Finally there have been several third-generation CAR T-cells engineered which carry two costimulatory sequences as well as further techniques to improve the persistence of CAR T ells (i.e., IL-2 injections), or in over-coming the tumor microenvironment’s immu-nosuppression (i.e., IL-12 and IFN-γ secretion as immunomodulating molecules) [79–81].

Currently, the use of CAR T-cell therapy has been directed toward antigens that have high expression on tumor cells and limited expression on normal cells due to concerns for life threaten-ing toxicity such as cytokine release syndrome (CRS). CRS has been associated with both BiTE antibodies and CAR T-cell therapy and encom-passes a constellation of inflammatory symptoms due to the cytokine release, mainly IL-10, IL-6 and IFN-γ, after T-cell engagement and prolif-eration [82]. Severe symptoms can include high fevers, hypotension, pulmonary edema, coagu-lopathy and multi-organ failure. Tocilizumab, a humanized anti-IL-6 receptor antibody, has been shown to lead to rapid improvement in patients with severe CRS [82]. Suicide gene sys-tems (i.e., HSV-tk and caspase-9) as a means to

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Table 1. Antibody-directed immunotherapy.

Immunotherapies Pediatric trials (phase)

Target Mechanism of action Current pediatric development

Monoclonal antibodies

Rituximab (Rituxan)

NCT01516580 [65] (III) NCT00066469 [66] (II) NCT01516567 [67] (II)

CD20 Type I mAb works mainly through CDC. Also has direct cytotoxicity via apoptosis and mild efficacy through ADCC

There are currently more than 30 open trials exploring the rituximab in treatment of lymphomas in pediatrics In pediatrics, it has been assessed in BL, NLPHL and PTLD

Ofatumumab (Arzerra)

NCT02419469 [26] (II) NCT02199184 [27] (II)

CD20 Type I mAb most effective mAb via CDC with ability to cause direct apoptosis and mild ADCC

Pediatric trails are assessing efficacy in B-LBL and BL

Obinutuzumab (Gazyva)

NCT02393157 [30] (II) CD20 Type II mAb works mainly through ADCC and has direct cytotoxicity by apoptosis with mild activity through CDC

A pediatric Phase II trial open for relapsed/refractory mature B NHL

Epratuzumab N/A CD22 Utilized in many forms such as a mAb, ADC and RIT

Has been assessed in relapsed/refractory pediatric B-ALL. There are no current pediatric lymphoma trials

Antibody–drug conjugates

Brentuximab Vedotin (Adcetris)

NCT01979536 [41] (II) NCT01780662 [42] (I/II) NCT02166463 [43] (III) NCT01492088 [68] (II)

CD30 ADC targeting the CD30 surface molecule linked to the microtubule disrupting agent monomethyl auristatin E

Has shown efficacy in adult ALCL and HL Currently being assessed in pediatric patients with newly diagnosed CD30+ ALCL, newly diagnosed high-risk HL, relapsed/refractory HL and relapsed/refractory ALCL

Inotuzumab Ozogamicin (CMC-544)

N/A CD22 ADC targeting the CD22 which is internalized and the linked agent calicheamicin leads to double stranded breaks

There are adult Phase I/II studies looking at CD22 NHL but no current pediatric trials

Moxetumomab pasudotox

NCT02227108 [49] (II) CD22 ADC targeting CD22 that delivers a recombinant immunotoxin that inhibits protein synthesis

One open Phase II pediatric B-cell ALL and lymphoblastic lymphoma

Polatuzumab Vedotin

N/A CD79b ADC targeting the CD79b surface molecule linked to the microtubule disrupting agent monomethyl auristatin E

There are adult Phase II trials assessing efficacy in DLBCL but no current pediatric trials

Radioimmunotherapy (antibody with radioisotope)

I131-tositumomab (Bexxar)

N/A CD20 Combined effects of a murine mAb (CD20) and radioisotope I131 in B-cell malignancies

No pediatric studies open

Y90-ibritumomab tiuxetan (Zevalin)

N/A CD20 Combined effects of a murine mAb (CD20) and radioisotope 90Y in B-cell malignancies

There was one Phase I pediatric trial assessing efficacy in NHL which was terminated

Radiolabelled anti-CD30 antibody: 131I-Ki-4

N/A CD30 Combined effects of a murine mAb (CD30) and radioisotope 131-iodine

Was efficacious in Hodgkin’s disease but led to severe hematologic toxicity limiting further trials

131I-F16SIP (Tenarad)

N/A Tenascin-C A1 domain

Form of RIT using a small immunoprotein (SIP) targeting the Tenascin-C isoform with an A1 domain largely found absent in regular tissue, but elevated in tumor tissue. A SIP is used for improved pharmacokinetics and is linked to radionucleide (131-iodine) for cytotoxic effect

Has been assessed in adult patients with chemo-refractory HL with objective response. No current pediatric trials

ADC: Antibody–drug conjugate; ADCC: Antibody-dependent cellular cytotoxicity; ALCL: Anaplastic large cell lymphoma; B-ALL: B-cell acute lymphoblastic leukemia; BL: Burkitt lymphoma; B-LBL: B-cell lymphoblastic lymphoma; CDC: Complement-dependent cytotoxicity; DLBCL, Diffuse large B-cell lymphoma; HL: Hodgkin lymphoma; mAb: Monoclonal antibody; NHL: Non-Hodgkin lyphoma; NLPHL: Nodular lymphocyte predominant Hodgkin lymphoma; PTLD: Post-transplant lymphoproliferative disease; RIT: Radioimmunotherapy.

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mitigate severe CRS in patients receiving CAR T-cell therapy, are currently being developed to become an off switch for the CAR T cells [83].

In a recent Phase I trial, 30 children and adults with relapsed/refractory precursor B-ALL, including 15 who had undergone prior allo-HCT, were treated with CAR T cells directed against CD19 [84]. A total of 90% of patients achieved a morphologic CR and 6-month EFS was 67% (95% CI: 51–88%). In addition to the CD19-engineered CAR, CAR T cells targeting CD20 have been developed. In a study by Wang and colleagues, seven adult patients with refrac-tory/advanced CD20-positive DLBCL received CD20 CAR T-cell therapy of which six of the seven patients had objective responses including one with originally bulky disease who was in a CR for 14 months at time of the report [85]. Delayed toxicities related to the CAR T-cell infusion were directly correlated with tumor burden and tumor-harboring sites in the patients and included CRS, tumor lysis, massive hem-orrhage of the alimentary tract and aggressive intrapulmonary inf lammation surrounding extranodal lesions. Additionally CAR T-cells have been developed to target the T-cell antigen CD5 which is present on approximately 80% of T-ALL and T-LL. These CD5 CAR T-cells have been shown to eliminate T-ALL and lym-phoma cell lines in vitro and inhibit T-ALL progression in mouse models [86]. Although CAR T-cell therapy can be highly effective, the robust response of CAR T cells has been associated with significant side effects as previ-ously mentioned [84,85]. Thus some have advo-cated for a debulking regimen prior to infusion of CAR T cells to minimize the toxicities that can accompany this immunotherapy, particu-larly when targeting lesions in special sites such as the lungs or alimentary tract where the tox-icities can be great [85]. In contrast to treating leukemia, treating lymphomas or solid tumors with CAR T-cell therapy can be more difficult and challenging as there are mechanical barri-ers, such as disorganized vasculature and stiff tissue, impaired lymphocyte trafficking to the tumor as well as the immunosuppressive tumor microenvironment [87]. The latter may be over-come by combining CAR T-cell therapy with immunomodulatory agents, including cytokines and checkpoint inhibitors, or by genetically engineering CAR T-cells to express or secrete immunomodulatory agents [88,89]. Multiple pediatric trials assessing the efficacy of CD19

CAR T-cell therapy exist. Although the majority of these studies are aimed at B-cell ALL there are a few which include B-cell NHL [90–92] as well as CD19-positive relapsed BL [90,93]. Currently there is an anti-CD22 CAR T-cell Phase I study open for pediatric patients with recurrent or refractory CD22-expressing B-cell malignan-cies [94], and an adult trial investigating the effi-cacy of CD20 CAR T cells in CD20-positive NHL (NCT01735604) [95].

It was recently reported that CAR T cells can be induced to undergo early burn out or exhaus-tion leading to reduced efficacy in tumor kill-ing and failure to persist in the patient for long term [96]. This appears to be determined by the choice of costimulatory domain chosen for the CAR where CD28 may accelerate T-cell exhaus-tion and 4-1BB reduce the process and therefore be associated with greater CAR T-cell efficacy. This finding provides some insight into the development of future CARs and mechanisms that may underlie treatment failure or CAR T-cell persistence in some patients compared with others.

●● Immune checkpoint blockadeIn lymphatic tissues, T cells receive activation signals through the T-cell receptor, the costimu-latory receptor CD28 and cytokine receptors. Activation of naïve or resting T cells results in the upregulation of CTLA-4, which has high affinity for the CD28 ligands B71 and B72. Ligation of CTLA-4 to these ligands counteracts the effects of CD28 and during the early T-cell antigen response phase this decreases T-cell effector function and prevents immune medi-ated collateral damage to normal tissues [97]. Similarly, in peripheral tissues, memory T cells express PD-1. Ligation of PD-1 recruits ship phosphatases, which dephosphorylate TCR signaling proteins resulting in decreased pro-liferation, cytotoxicity, cytokine production, expression of survival proteins and increased susceptibility to apoptosis [98]. Thus similar to CTLA-4 ligation, PD-1 ligation protects periph-eral tissues from T-cell-mediated collateral dam-age during inflammatory responses by decreas-ing T-cell effector function. Thus the term immune checkpoints refers to a class of proteins responsible for maintaining self-tolerance and modulating physiologic immune responses in peripheral tissues in order to minimize collateral tissue damage [99]. However, the PD-1 receptor-ligand interaction is a major pathway used by

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tumors to suppress the host immune response and therefore inhibition by checkpoint inhibi-tors may restore the T-cell cytotoxicity against tumor cells [100].

These immune checkpoints are now known to be one mechanism by which tumors evade the immune system. CTLA-4 activation has been shown to downregulate the immune response against a variety of tumors [99]. As well, PD-1 is highly expressed by tumor infiltrating lympho-cytes and PD-L1/PD-L2 is constitutively over-expressed on a number of solid tumors and lym-phomas. PD-1 interacts with the PD-L1/PD-L2 ligands that are upregulated on tumor cells and leads to downregulation in the T-cell-killing response [100,101]. Checkpoint inhibitors are antibodies that block CTLA-4, PD-1 or PD-1L and break immune tolerance. Pediatric lympho-mas which are known to express PD-1 pathway receptors include ALCL (PD-L1), DLBCL (PD-L1), HL (PD-L1 and PD-L2) and PMBCL (PD-L2) [102–104].

Antibodies directed against CTLA-4 were the first class of immune checkpoint inhibitors to receive FDA approval. Ipilmumab is a fully humanized IgG1 antibody to CTLA-4 and is the first agent in the past 30 years to prolong OS in adults with unresectable stage III and stage IV malignant melanoma. Since then, several clini-cal trials have tested it in a variety of tumors including relapsed/refractory HL and NHL in adults. In a Phase I study of 18 adult patients with B-cell NHL, two clinical responses were observed; one CR in a patient with DLBCL and a PR in a patient with follicular lymphoma [105]. The more common AEs included diarrhea, headache, abdominal pain, anorexia, fatigue, neutropenia and thrombocytopenia. Bashey and colleagues reported a Phase I study using ipilmumab in the setting of relapse post allo-HCT for hematologic malignancies [106]. The study enrolled 29 adult patients of which 48% (n = 14) had HL and reported an ORR of 10% comprised of two CRs in patients with HL and a PR in a patient with mantle cell lymphoma. Although no DLT was observed, AEs grade ≥3 included anemia, thrombocytopenia, neutrope-nia/fever, elevated AST/ALT, dyspnea, mucosi-tis, arthritis, hyperthyroidism, infection, hip fracture and pneumonitis.

Nivolumab is a PD-1 blocking IgG4 antibody that has shown tolerability and activity in HL. Evidence supports that the Reed-Sternberg cell, diagnostic of HL, exploits the PD-1 pathway

as a means of evading immune detection [104]. In a Phase I trial of 23 adult patients with relapsed/refractory HL, 20 patients achieved an objective response (87%) including four (17%) who reported a CR [104]. Of the 23 patients, 12 discontinued treatment for disease progres-sion (n = 4), toxicity (n = 2) or proceeding to allo-HCT (n = 6). Grade ≥3 AEs included lymphopenia, elevated lipase, stomatitis, pan-creatitis and myelodysplastic syndrome. Based on the results from this study in heavily pre-treated adults with HL, the US FDA granted nivolumab breakthrough therapy designation for adult patients with HL who failed prior ASCR and brentuximab vedotin. The COG is currently investigating nivolumab in a Phase I/II study in children, adolescents and young adults with recurrent or refractory solid tumors of which HL and NHL are included (NCT02304458) [107].

Pidilizumab is another PD-1 blocking anti-body (IgG-1 mAb) that has been used in adult NHL. In an international Phase II study assess-ing pidilizumab in 35 adult patients with DLBCL who had measurable disease following ASCR, 12 patients achieved a CR and four a PR for an ORR of 51% [108]. The most common AEs grade ≥3 were neutropenia (19%) and thrombocytopenia (8%). A third example of a PD-1 blocking anti-body is pembrolizumab, which also has reported encouraging response rates in adult lymphoma similar to both nivolumab and pidilizumab. Pembrolizumab is being tested in a Phase I study of 15 adult patients with relapsed/refractory HL and reported preliminary results with an ORR of 53% including three patients with a CR (NCT01953692) [109,110]. Pembrolizumab is also undergoing a Phase I/II study in pedi-atric patients (ages 6 months to 17 years) with relapsed/refractory lymphoma as well as mela-noma and solid tumors (NCT02332668) [111]. See Table 2 for a more comprehensive list of immunomodulatory therapies.

Although not considered part of the immune checkpoint cascade, B-cell receptor (BCR) sign-aling plays an important role in the pathogenesis of B-cell malignancies. Downstream of the BCR is the Bruton tyrosine kinase (BTK) receptor, a signal-transduction kinase responsible for BCR signaling and cell development [114]. Ibrutinib is an orally administered small molecule BTK inhibitor whose mechanism of action consists of covalently binding to a cysteine residue in the BTK active site resulting in sustained inhi-bition of kinase activity [115]. In adult studies,

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Table 2. Immunomodulating agents.

Immunotherapies Pediatric trials (phase) Target Mechanism of action Current pediatric development

Bispecific antibodies

Blinatumomab (Blincyto)

N/A CD19 T-cell recruitment with dual specific antibody for CD3 and CD19

Currently undergoing Phase III assessment for the treatment of Philadelphia chromosome-negative relapsed/refractory B-ALL. There are no current pediatric lymphoma trials

CD20-TDB N/A CD20 T-cell recruitment with dual specific antibody for CD3 and CD20

Preclinical but shown effective in vitro and in vivo studies

AFM13 N/A CD30 NK cell recruitment via CD16A targeting CD30 cells

Currently being assessed only in adult HL

Cellular therapy

CAR T-cell CTL

NCT02259556 [112] (I/II) NCT00840853 [90] (I) NCT02443831 [93] (I)

CD30

Engineered T cells that recognize specific tumor targeted antigens triggering tumor killing

A) Assessing patients 16 years and older in Phase I/II trials with CD30+ lymphomas

NCT01593696 [91] (I) B) CD30 CAR T-cells are in Phase I/II studies for patients >16 years old who have relapsed after transplant

NCT01626495 [92] (I/II) C) CD20 CAR T cells being assessed in adult CD20+ NHL

NCT02315612 [94] (I) D) CD19 CAR T cells being assessed in CD19+ leukemia and lymphoma

E) CD22 CAR T cells being assessed in CD22+ ALL and lymphoma

CTLs (EBV specific)

NCT02057445 [72] (I) NCT01555892 [73] (I) NCT01636388 [77] (II) NCT01956084 [75] (I) NCT01447056 [76] (I) NCT02287311 [74] (I)

EBV specific antigens

CTL against EBV antigens are expanded in vivo with irradiated B-cells and response against EBV driven malignancy

Current pediatric Phase I/II trials assessing efficacy in EBV + recurrent/refractory lymphomas including HL and NHL

CTLs (MAGE-A4) N/A Tumor antigen-specific T-cell

CTL against Mage-A4 (protein required for tumor anti-apoptosis by interacting with p53). Effects are upregulated with decitabine

Currently adult Phase I trials where several different tumor associated antigens (TAAs) are being targeted in both relapsed/refractory HL or NHL

Checkpoint inhibitors

PD-1 Ab (nivolumab)

NCT02304458 [107] (I/II) PD-1 Inhibits PD-1 a receptor that is upregulated on Reed-Sternberg cells that down regulates antitumor T-cells and leads to their apoptosis

Currently being assessed with recurrent/refractory solid tumors including NHL and HL

PD-1 Ab (pidilizumab)

N/A Has been assessed in adult DLBCL but there are no current pediatric lymphoma trials

PD-1 Ab (pembrolizumab)

NCT02332668 [111] (I/II) Currently undergoing a Phase I/II study in pediatric patients with relapsed/refractory lymphoma and other solid tumors

CTLA-4 Ab (ipilimumab)

NCT01445379 [113] (I) CTLA-4 CTLA-4 is an opposing inhibitory signal that increases the threshold for T-cell activation. Inhibiting CTLA-4 with specific monoclonal antibodies enhances T-cell anti-tumor responses

Assessing efficacy and safety in pediatric patients with relapsed/refractory lymphoma along with other solid tumors

ALL: Acute lymphoblastic leukemia; CAR: Chimeric antigen receptor; CTL: Cytotoxic T lymphocyte; DLBCL: Diffuse large B-cell lymphoma; EBV: Ebstein–Barr virus; HL: Hodgkin lymphoma; NK: Natural killer; NHL: Non-Hodgkin lymphoma.

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ibrutinib has been tested in DLBCL and other B-cell malignancies with varying success [116–

118]. In a Phase I study of 56 adult patients with relapsed/refractory B-cell lymphoma and chronic lymphocytic leukemia, ibrutinib as a single agent was well tolerated and achieved an ORR of 60% including a CR in 9/56 (16%) patients [119]. AEs grades ≥3 included diarrhea, nausea/vomiting, decreased appetite, fatigue, pain and fever. Although there is no current pedi-atric data published using ibrutinib, a Phase II study in patients with B-cell NHL 16 years and older is planning to open (NCT02436707) [120].

Conclusion & future perspectiveAlthough treatment decisions for recurrent or refractory lymphoma have been limited in chil-dren, immunotherapy is becoming a promising option. As early-phase pediatric trials testing a variety of immunotherapies continue in their

early stages, the results of these trials in adults with high-risk lymphoma continue to show promise. If the pediatric studies prove toler-able and report similar response rates as seen in adults, then future pediatric studies may bring these exciting agents into the upfront setting for children and young adults with newly diagnosed HL and NHL.

Financial & competing interests disclosureThis work was supported by the Midwest Athletes Against Cancer (MACC) Fund and the Medical College of Wisconsin High-Risk Hematologic Malignancy Program. The authors have no other relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

EXECUTivE SUMMARYRelapsed/refractory pediatric lymphomas have poor outcomes

● Although cure rates for the majority of children with Hodgkin lymphoma/non-Hodgkin lymphoma are excellent using traditional chemotherapies, patients with relapsed or refractory disease have dismal outcomes.

● Long-term treatment related toxicity remains a significant burden in our survivor population.

● Immunotherapies which employ a targeted and effective approach are becoming an attractive option when considering salvage therapy for these young patients.

Immunotherapy has expanded considerably in pediatric lymphoma

● Until recently, monoclonal antibodies (mAbs) had been the primary immunotherapy used in childhood lymphoma.

● The traditional naked mAb has since been expanded to include antibody–drug conjugates and radioimmunotherapy which has enhanced the tumor response rate for these patients.

● The immunotherapy armamentarium available to pediatric lymphoma now goes beyond the original mAbs to include antibody–drug conjugates, Radioimmunotherapy, cytotoxic T lymphocytes, chimeric antigen receptor T-cell therapy, bispecific T-cell engager and immune checkpoint inhibitors.

● Increasing the number of early-phase trials for pediatric lymphoma with developing immunotherapies should continue with the ultimate goal of curing more children and minimizing the treatment related toxicities that have been so strongly associated with this disease group.

ReferencesPapers of special note have been highlighted as: • of interest; •• of considerable interest

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