New Strategies in Bladder Cancer: a Second Coming for Immunotherapy Ali Ghasemzadeh1, Trinity J

Published OnlineFirst December 18, 2015; DOI: 10.1158/1078-0432.CCR-15-1135

CCR New Strategies Clinical Cancer Research New Strategies in Bladder Cancer: A Second Coming for Immunotherapy Ali Ghasemzadeh1, Trinity J. Bivalacqua1,2,3, Noah M. Hahn1,3, and Charles G. Drake1,3

Abstract

Urothelial bladder cancer (UBC) remains one of the most prolonged survival for patients with several different can- common and deadly cancers worldwide, and platinum- cers, including UBC. In this review, we discuss new ﬁndings based chemotherapy, which has been the standard-of- in bladder cancer immunotherapy, including recent suc- care in metastatic bladder cancer, has had limited success cesses with immune checkpoint blockade. We also discuss in improving outcomes for patients. The recent develop- therapeutic cancer vaccines and highlight several additional ment and translation of therapeutic strategies aimed at immunotherapy modalities in early stages of development. harnessing the immune system have led to durable and Clin Cancer Res; 22(4); 1–9. 2015 AACR.

Background mutational burden (11). Mutations can provide neoantigens that can be recognized by the immune system (12). Indeed, a higher Urothelial bladder cancer (UBC) is the sixth most common mutational load has been correlated with better response rates to cancer in the United States, with 74,690 cases diagnosed and immune checkpoint blockade in patients with lung cancer and 15,580 deaths in the year 2014. Similar to many cancers, it is a melanoma (13, 14). These observations and the historically poor disease of the elderly, with a median age of 73 years at diagnosis performance of current therapies in UBC provide a unique oppor- (1). Risk factors for the development of bladder cancer include tunity for the use of immunotherapy. Although a number of cigarette smoking and exposure to cyclic chemicals, dyes, rubbers, excellent reviews have recently described emerging data in tumor textiles, and paints (2). Smoking is the number one environmen- immunology (15–17), the focus of this review is to more specif- tal risk factor and may be associated with the relatively high ically highlight recent advances in immunotherapy for UBC. mutation rate noted in UBC (3, 4). Consequently, this review first provides a brief overview of con- Approximately 70% of bladder cancers are diagnosed when ventional immunotherapy for UBC [bacillus Calmette–Guerin they are non–muscle-invasive (NMIBC; ref. 5). For these (BCG)], and then discusses immune checkpoint blockade as well tumors, transurethral resection of bladder tumor is the standard as agonists and vaccines intended to initiate an antitumor of care and aids in proper staging of the disease. For more immune response. advanced (muscle-invasive disease), the last major therapeutic BCG is an attenuated form of the bovine tuberculosis bacterium advance was the introduction of platinum-based chemotherapy Mycobacterium bovis. The first clinical trial of BCG in bladder cancer almost 30 years ago (6). For patients with advanced UBC, in 1980 showed a 20% reduction in recurrence rate (18). Despite treatment options are limited and often ineffective, and out- its long history, the mechanism of action of BCG is not yet fully comes remain poor (7, 8). Furthermore, for patients for whom understood and is beyond the scope of this article but has recently first-line chemotherapy is not successful, no clearly defined been reviewed (19, 20). Clinically, randomized controlled trials second-line option exists and none have definitively been (RCT) have shown that in NMIBC, BCG immunotherapy reduces shown to prolong overall survival (OS; refs. 9, 10). rates of recurrence and progression while positively affecting Recent understanding of the genomic alterations underlying mortality. RCTs comparing monthly, quarterly, and biannual bladder cancer have not yet led to new targeted therapies, but BCG maintenance showed no sign of increased efficacy compared these insights show UBC to be a cancer with high somatic with induction BCG alone (21–23); however, the 3-week maintenance schedule of the Southwest Oncology Group demonstrated significant benefit with a 76.8-month recurrence-free survival 1 Department of Oncology, Johns Hopkins Sidney Kimmel Compre- duration (RFS) in the maintenance arm of the study compared hensive Cancer Center, Baltimore, Maryland. 2Department of Surgery, Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Balti- with 35.7 months with induction therapy alone (24). The 5-year more, Maryland. 3The Brady Urological Institute, Johns Hopkins Sid- survival rate was 78% with induction therapy alone compared ney Kimmel Comprehensive Cancer Center, Baltimore, Maryland. with 83% with maintenance. Thus, only 3-week BCG mainte- Corresponding Author: Charles G. Drake, Departments of Oncology, Immunol- nance has been shown in RCTs to reduce disease progression and ogy, and Urology, Johns Hopkins University School of Medicine, 1650 Orleans improve overall and disease-specific mortality (24, 25). Three- Street, CRB 4M51, Baltimore, MD 21287. Phone: 410-614-3616; Fax: 443-287- week maintenance BCG has also been shown to be superior to 4653; E-mail: [email protected] maintenance with epirubicin chemotherapy and combination doi: 10.1158/1078-0432.CCR-15-1135 treatment with epirubicin and IFNa (25, 26). Intravesical BCG 2015 American Association for Cancer Research. immunotherapy remains the gold standard for the treatment of

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NMIBC, and exciting possibilities exist for improving outcomes non–small cell lung cancer (NSCLC), renal cell carcinoma, and in patients with superficial disease by combining BCG with bladdercancer(30–33). other immune-modulatory therapies (Table 1). CTLA-4: the first immune checkpoint. CTL-associated antigen 4 (CTLA4) was the first immune checkpoint for which blockade was On The Horizon shown to enhance antitumor immunity (34). Because CTLA-4 Immune checkpoint blockade functions by dampening T-cell activation, blocking of CTLA-4 The genetic alterations that occur during carcinogenesis enhances T-cell activation. This process is complex; normally, T- provide numerous antigenic targets that can be recognized by cell activation requires two signals; the first is a signal delivered the immune system (12). However, cells of the adaptive when the T-cell receptor recognizes its specific antigen. A second immune system are often inhibited by pathways that are signal is required for full T-cell activation; this signal 2 is delivered dysregulated in tumors and act to suppress their effector func- when B7 molecules on a mature dendritic cell (DC) bind to CD28 tions (27). Over the past 20 years, we have gained greater on the partially activated T cell. T-cell expression of CTLA-4 hijacks understanding of these pathways, which are mediated by cell this process, binding with high affinity to B7 molecules and surface molecules collectively termed "immune checkpoints." preventing the T cell from receiving signal 2 (Fig. 1; ref. 35). In These entities represent a group of functionally related but patients, administration of anti-CTLA-4 leads to increased T-cell biologically distinct cell surface receptors with important phys- activation, a significant rate of objective responses, and a consid- iologic roles in maintaining self-tolerance, modulating the erable rate of immune-related adverse events (irAE; refs. 36, 37). immune response to pathogens, and preventing autoimmunity In that regard, the rate of irAEs induced by CTLA-4 blockade is (28, 29). Blocking antibodies targeting immune checkpoints higher than that observed in patients treated with agents that have shown promise in several cancers, including melanoma, block the checkpoint mediated by the interaction between PD-1

Table 1. Selected immunotherapy trials in UBC Trial ID Immunotherapy Phase n Primary endpoint Remarks CTLA-4 NCT00362713 Ipilimumab I 12 Safety Neoadjuvant therapy; completed NCT01524991 Ipilimumab II 36 OS In combination with gemcitabine and cisplatin; ongoing

PD-1 NCT01848834 Pembrolizumab I 297 Safety response rate Bladder cancer cohort in a large phase I trial; ongoing NCT02324582 Pembrolizumab I 15 Safety In combination with BCG vaccine; ongoing NCT01928394 Nivolumab I/II 410 Objective response rate In combination with ipilimumab; ongoing NCT02351739 Pembrolizumab II 74 Objective response rate In combination with ACP-196 (Btk inhibitor); ongoing NCT02256436 Pembrolizumab III 470 OS In comparison to: paclitaxel, vinﬂunine, docetaxel in patients with disease recurrence or progression following platinum-based therapy; ongoing

PD-L1 NCT01375842 Atezolizumab I 344 Dose-limiting toxicities Bladder cancer patients accepted in a large phase I trial; ongoing NCT02108652 Atezolizumab II 439 Objective response rate Potential registration trial; ongoing NCT02451423 Atezolizumab II 42 Change in T-cell count Neoadjuvant therapy in BCG-refractory NMIBC or MIBC prior to Pathologic T0 rate cystectomy; ongoing Pathologic absence of disease in the bladder NCT02450331 Atezolizumab III 440 DFS Patients with MIBC at high risk of recurrence following cystectomy; ongoing NCT02302807 Atezolizumab III 767 OS In comparison to: paclitaxel, vinﬂunine, docetaxel in patients who have progressed during or following platinum-based therapy; ongoing

B7-H3 NCT01391143 MGA271 I 151 Safety In patients with refractory cancer; ongoing

Vaccines NCT02010203 Vesigenurtacel-L I/II 84 Phase I: safety In combination with BCG vaccine; ongoing Phase II: 1-year DFS NCT01353222 Lapuleucel-T II 180 OS Ongoing

CSF1R NCT02452424 PLX3397 I/II 400 Safety in combination with In combination with pembrolizumab; ongoing pembrolizumab IDO1 NCT02178722 INCB024360 I/II 120 Phase I: safety In combination with pembrolizumab; ongoing Phase II: PFS Abbreviations: DFS, disease-free survival; OS, overall survival; PFS, progression-free survival.

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(PDCD1) and its ligands (38). The mechanistic basis for the tive biomarker for treatment response (33). Overall, 55% of difference in side-effect profiles is not completely understood. patients showed a reduction in tumor burden by RECIST However, one potential explanation is that CTLA-4 is highly criteria and responses were rapid, occurring at a median of expressed on regulatory T cells (39), which play an important 42 days. On the basis of these data, atezolizumab was granted role in maintaining peripheral tolerance (40, 41) and which are Breakthrough Therapy Designation by the FDA in May 2014. A present in abundance in multiple tissue types including the skin, single-arm phase II trial of atezolizumab evaluating the efficacy gut, liver, and others. PD-L1 (CD274), by contrast, is not consti- and safety in patients with locally advanced and metastatic tutively expressed; expression is limited to tumors and to areas of UBC who are treatment na€ve and ineligible for platinum-based active inflammation, where the PD-1/PD-L1 interaction likely chemotherapy or who have progressed on a platinum-based serves to downmodulate an immune response during the effector regimen has accrued (NCT02108652), although efficacy data phase (42). are not yet available. Anti-PD-1 (pembrolizumab; Merck) has In addition to being FDA approved for melanoma (43), CTLA-4 also been evaluated in a relatively small cohort (N ¼ 33) of blockade has also been evaluated in a small number of bladder patients with UBC. The ORR was similar to that for PD-L1 cancer patients. In a study of 12 patients with localized UBC who blockade at 28% (56). Median PFS and OS durations for UBC received neoadjuvant ipilimumab at a dose of either 3 or 10 mg/kg patients were 2 months and 12.7 months, respectively. As prior to cystectomy, the drug was shown to be safe and led to an expected, PD-L1 expression correlated with response; patients increase in CD4 and CD8 T cells in both tumor and blood (44). A in whom tumor and tumor-infiltrating immune cells were PD- second phase II trial recently completed accrual of 36 patients; this L1 negative had a 0% ORR in contrast to patients that were PD- single-armed study (NCT01524991) treats patients with standard L1 positive who had a 29% ORR. A second-line phase III study first-line chemotherapy (gemcitabine and cisplatin) in addition to comparing pembrolizumab treatment to paclitaxel, docetaxel, ipilimumab (Table 1). The primary endpoint is 1-year survival. or vinflunine in patients with metastatic or unresectable UBC is Although efficacy data are not yet available, interim analyses now recruiting patients (NCT02256436). It should be noted show an on-treatment increase in CD4 and CD8 T cells with an that the favorable safety profile of PD-1 and PD-L1 blocking enhanced inflammatory cytokine signature including IL2, IL12, antibodies may play an important role in their development in and granulocyte-macrophage colony-stimulating factor (GM- UBC; approximately 48% of patients forgo chemotherapy in CSF; ref. 45). Although CTLA-4 is clearly an important immune the second-line setting due to its relatively limited clinical checkpoint, moving anti–CTLA-4 into earlier-stage UBC or into benefit(57). combination regimens may be limited by its toxicity profile (46). In UBC, PD-L1 status is emerging as a possible predictive biomarker. However, there has been great variation in the assays PD-1/PD-L1: the next generation checkpoint. The programed used to measure PD-L1 status to date (58). Sources of variation death-1 (PDCD1, PD-1) pathway has emerged as an exciting have included the use of at least four different PD-L1 antibody therapeutic target in cancer therapy (27). Although CTLA-4 clones; staining positivity cutoffs ranging from 1% to 10%; serves to inhibit T cells at the initial activation step, PD-1 and inclusion of tumor cells, immune cells, or both in the appears to function by downregulating ongoing immune PD-L1 status. The standardization of antibodies, cutoffs, biopsy responses at sites of infection, that is, either in the periphery types, and analysis techniques will greatly aid in achieving a or in the tumor parenchyma (47). PD-1 is expressed during better understanding of the role of this dynamic biomarker in initial T-cell activation, and remains upregulated on exhausted, predicting responses to therapy. nonfunctional cells (48), and signaling occurs through binding Patients with PD-L1–positive tumor and tumor-infiltrating of its major ligand partners PD-L1 (CD274 or B7-H1) and PD- immune cells had response rates ranging from 29% to 43%. Thus, L2 (CD273 or B7-DC; Fig. 1). Although there are similarities in among patients with high PD-L1 expression, a sizeable propor- the signaling of PD-1 and CTLA-4, clinical data show these tion did not respond to treatment. An understanding of the pathways play nonredundant roles in inhibiting immune determinants of response to PD-1/PD-L1 blockade is critically responses (49, 50). important for increasing the proportion of responding patients. Multiple studies showed that UBC expresses PD-L1 at similar Interactions among members of the tumor microenvironment, levels to other tumors (approximately 10%–20% of tumor cells) including tumor-associated macrophages (TAM), vascular endo- with increased levels of PD-L1 expression seen in more advanced thelial cells, cancer-associated fibroblasts, and immunosuppres- and metastatic tumors compared with early-stage disease (33, 51– sive metabolites such as kynurenine can all play an important role 55). Moreover, increased PD-L1 expression in these tumors has in dictating the level of T-cell infiltration in the tumor (17). been associated with reduced OS and RFS following cystectomy. Tumor-intrinsic mutations in the b-catenin pathway have been These data support the notion that bladder tumors may evade the demonstrated to mediate cancer immune evasion and resistance immune system by upregulating PD-L1 expression. In the past to anti–PD-L1 therapy (59). year, exciting data have emerged from clinical trials of agents that block the PD-1/PD-L1 interaction. In a cohort of UBC patients in a B7-H3: highly expressed, but less clear in function. B7-H3 (CD276) phase I study, the activity of MPDL3280A (atezolizumab; Gen- is a member of the B7 family of cell surface receptors whose entech, Inc.), a humanized IgG1 antibody against PD-L1, was expression can be induced on activated DCs, macrophages, T cells, examined. The overall response rate (ORR) was 35%, and the drug B cells, and natural killer (NK) cells (Fig. 1; ref. 60). Its role in was well tolerated, with no treatment-related deaths and a 5% rate antitumor immunity is complex, with both stimulatory and of grade 3–4 irAEs. The ORR was 43% in patients who were IC2/3 inhibitory functions reported (61, 62). The receptor for B7-H3 for PD-L1 expression on immune cells, whereas patients with remains unidentified as well. Nevertheless, targeting B7-H3 may lower levels of PD-L1 expression (IC0/1) had an ORR of 11%, be particularly important in UBC as reports have shown between suggesting that PD-L1 expression might serve as a weakly predic- 58% and 70% of tumors express this molecule (53, 54). An IgG1

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A Immature dendritic cell Activation Activated dendritic cell Suppression

Agonist antibodies MEDI6469 (anti-OX40) BMS-663513/PF05082566 (anti–4-1BB) CP-870,893 (anti-CD40)

Vaccines OX40L BCG 4-1BBL Vesigenurtacel-L CD8 T cell Lapuleucel-T OX40 4-1BB

B7-H3 (ligand unknown) CD40L TCR Lymph node

CD40 CD28 CTLA-4 MHC I CD80/86 Monoclonal antibodies Ipilimumab (anti–CTLA-4) MGA271 (anti–B7-H3)

B B7-H3 (ligand unknown) CD8 T cell

MHC I TCR Monoclonal antibodies Nivolumab (anti–PD-1) Pembrolizumab (anti–PD-1) Atezolizumab (anti–PD-L1) PD-L1 MGA271 (anti–B7-H3) PD-1

PD-L1 Tumor cell PD-L2 CSF1 Tumor microenvironment

CSF1R IL34 M2 (suppressive) IDO1 Small-molecule inhibitors PLX3397 (CSF1R inhibitor) FPA008 (anti-CSF1R antibody) Tryptophan Metabolites Tumor-associated macrophage Emactuzumab (anti-CSF1R antibody) INCB024360 (IDO1 inhibitor) GDC919 (IDO1 inhibitor)

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antibody against B7-H3 has been developed (MGA271, Macro- cells with proprietary fusion protein that combines a tumor genics, Inc.; ref. 63), and is now in a phase I clinical trial enrolling antigen (in this case HER2/neu) with GM-CSF, which serves patients with a variety of advanced cancers including UBC to mature the peripheral blood mononuclear cells into antigen- (NCT01391143; Table 1). presenting DCs. In a phase I study of 18 patients with HER2/ neu–expressing tumors, immune responses to the target anti- Cancer vaccines gen were noted, the therapy was well tolerated, and 2 of 18 Cancer vaccines aim to initiate T-cell responses against patients experienced stable disease lasting >48 weeks (69). This tumor antigens by inducing activated antigen-presenting cells vaccine is now in a phase II trial evaluating survival, safety, fi and immune responses in the adjuvant setting in patients with (APC) that express a tumor-associated or speci c antigens (64). þ Activated APCs then drive the proliferation and function of high-risk HER2 UBC (NCT01353222; Table 1). specific T cells, which have the potential to mediate tumor cell lysis. Historically, vaccine approaches to cancer treatment have Agonist antibodies: activating immune cells via been limited by T-cell exhaustion, an altered differentiation costimulatory molecules state which manifests as a progressive and hierarchical loss In addition to immune checkpoint molecules, whose engage- of effector functions and upregulation of multiple inhibitory ment downregulates T-cell function, a number of molecules checkpoint molecules on the T-cellsurface(48).Thesuccessof provide a positive signal to T cells, and the ultimate outcome of checkpoint molecule inhibitors in reversing T-cell exhaustion a T cell's interaction with an APC involves an integration of has important implications for the use of therapeutic cancer both the positive and negative signals present during that vaccines in the clinic. Several vaccines are under evaluation in interaction (Fig. 1). Although still in early stages of develop- UBC. In this section, we highlight two recent approaches. ment, agonist antibodies that activate immune cells have tre- mendous potential in UBC and are introduced here with an eye A tumor cell–based vaccine: vesigenurtacel-L. Vesigenurtacel-L toward future advancements. (Heat Biologics, Inc.) is an example of a cell-based vaccine; these types of vaccines have the theoretical advantage of presenting a OX40 on T cells. A member of the TNF receptor superfamily, number of tumor-associated antigens simultaneously (65). To OX40 (TNFRSF4, CD134) is a cell-surface molecule expressed generate this vaccine, an allogeneic bladder cancer cell line was when either CD4 or CD8 T cells recognize their specificantigen modified to secrete the endoplasmic reticulum chaperone protein (70). Engagement between OX40 on a T cell and OX40 ligand gp96 (HSP90B1). gp96 has two immunologically relevant bio- on an APC provides a powerful costimulatory signal to the logic functions: (i) it chaperones peptides generated by intracel- T cell (71). Not only does OX40 provide a stimulatory signal lular proteosome degradation into class I MHC; and (ii) gp96 to effector T cells, it may also provide an inhibitory signal released from cells can function as a danger-associated molecular to regulatory T cells (ref. 72; Fig. 1). In a phase I study of a pattern and activate DCs by binding to TLR-2 and TLR-4 (66). murine anti-OX40 (MEDI6469; Medimmune) in patients with Mechanistically, this vaccine is thought to function when gp96 advanced cancer, the drug had an acceptable safety profile and released from dying vaccine cells binds to CD91 on host APCs, induced the regression of at least one metastatic lesion in 40% triggering endocytosis, and import of tumor cell antigens, of patients (73). This murine anti-OX40 is now in clinical trials which are subsequently presented to CD8 T cells in the context in combination with chemotherapy and radiotherapy in pros- of class I MHC. A first-in-human trial of a similar vaccine tate cancer (NCT01303705) and breast cancer (NCT01862900) in NSCLC demonstrated this approach to be safe, with 7 of and in combination with anti–CTLA-4 in metastatic melanoma 18 patients showing stabilized tumor growth though none dis- (NCT01689870). A second anti-OX40 antibody (MOXR0916; played an objective response (67). A phase I/II trial in UBC Genentech, Inc.) is undergoing phase I testing in patients with (NCT02010203) has recently started accruing patients. After an advanced cancer (NCT02219724). initial safety run-in, this combination trial will quantify 1-year disease-free survival in patients with earlier-stage disease 4-1BB on T cells. Like OX40, 4-1BB (TNFRSF9) is also a member of (NMIBC) treated with BCG plus vesigenurtacel-L (Table 1). the TNF receptor superfamily, and is expressed on NK cells and activated T cells (71). Its ligand, 4-1BBL (TNFSF9), is expressed on A DC-based vaccine: lapuleucel-T. As vaccine responses are driven activated APCs, and its engagement by 4-1BB leads to increased by DCs, the most potent APC, one approach to cancer immu- proliferation and expression of antiapoptotic molecules in T cells notherapy is to generate DCs ex vivo,loadthemwithatumor- (Fig. 1). In an initial phase I study, an agonist 4-1BB antibody associated antigen, and then administer the cells to patients (BMS-663513, urelumab; Bristol-Myers Squibb) was well toler- (68). Lapuleucel-T (Dendreon) is an example of this approach ated in patients with metastatic melanoma, and while only 6% in UBC; the vaccine is prepared by isolating peripheral blood of patients had a partial response, 17% of patients showed monocytes from an individual patient, then culturing those stable disease at 6 months (74). On the basis of these results, a

Figure 1. Mechanism of action of selected immunotherapies in UBC. A, representation of cellular interactions within the lymph node. Adaptive immune responses initiate at the lymph node through the interaction of T cells and DCs. Vaccines initiate an immune response by providing target antigens to DCs and triggering their activation. Activated DCs in turn present antigen as well as costimulatory molecules and immune checkpoints to T cells. These molecules shape the quality and magnitude of the T-cell response. B, in the tumor microenvironment, T cells encounter cognate antigen and can cause tumor lysis. However, tumors often express inhibitory immune checkpoint molecules such as PD-L1 to inhibit T-cell responses. In addition to the tumor cells themselves, TAMs can also inhibit T-cell responses through a variety of mechanisms, including immune checkpoint expression and metabolic inhibition.

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phase II trial was launched but patients experienced severe hep- prostate cancer (NCT01560923). NLG919 is now in phase I trials, atitis and the trial was discontinued (75). A different 4-1BB including a trial in combination with atezolizumab that includes antibody, PF05082566 (Pfizer Inc.) is currently being investigated UBC patients (NCT02471846; Table 1). in three phase I trials in combination with pembrolizumab (NCT02179918) or anti-CCR4 (NCT02444793; mogamulizu- Combination therapy mab, Kyowa Hakko Kirin) in advanced solid tumors, and ritux- Checkpoint blockade will likely change the shape of the UBC imab for Hodgkin lymphoma (NCT01307267). Urelumab treatment landscape in the very near future, but it is clear from has also reentered clinical testing and is now being investigated existing data that the majority of patients will not respond in multiple phase I and II clinical trials in a variety of hema- to monotherapy. Several rational strategies to address that tologic and solid malignancies. issue are emerging. First, based on exciting clinical data in melanoma (49) and kidney cancer (85), combined checkpoint CD40 on APCs. Unlike OX40 and 4-1BB, CD40 is predominantly blockade is being evaluated in a variety of tumor types includ- expressed on APCs, and ligation of CD40 by its ligand CD40L, ing UBC. A second strategy involves inducing a tumor-specific which is expressed on CD4 T cells, leads to stimulation immune response via vaccination or intratumoral injection and maturation of DCs (Fig. 1; ref. 76). Consequently, this is an of immune-activating agents, including viral vectors (86). In indirect mechanism by which CD8 T-cell activation can be UBC, a phase II trial of intravesical adenoviral-mediated IFNa achieved (77). In a phase I study in patients with stage III gene therapy recently reported 10 of 34 patients experiencing a and IV solid tumors, CP-870,893, an anti-CD40 agonist antibody, complete response at 12 months (87). This trial is currently was well tolerated with 14% of patients showing a partial ongoing. It should be noted, however, that an influx of acti- response (78). Trials evaluating CP870,893 in combination vated IFNg-secreting T cells into a tumor is generally accom- with anti–CTLA-4 (NCT01103635), and paclitaxel and carbo- panied by an upregulation of PD-L1 on the tumor cells, a platin (NCT00607048) have been recently completed and process termed "adaptive immune resistance" (88) that sug- future trials are anticipated. gests that vaccines might be most effective when combined with a PD-1/PD-L1 blocking agent. Clinical trials are currently under Targeting the tumor microenvironment in UBC wayinUBCtestingcombinationsofcheckpointblockadeand CSF1R. CSF1R (CSF1R) is a cell surface receptor expressed chemotherapeutics, vaccine approaches, tyrosine kinase inhi- predominantly on macrophages and monocytes (79). This bitors, tumor microenvironment and myeloid cell targeting agent may be important in UBC because macrophages exist therapies, and metabolic enhancement strategies (Table 1). on a continuum from an M1 (inflammatory) to an M2 (pro- Moving forward, the design of such strategies will require a tumorigenic) phenotype and the presence of M2 macrophages thorough understanding of the determinants of the tumor in the UBC stroma has been associated with BCG immuno- immune environment in UBC. therapy failure (80). CSF1 ligation promotes the skewing of macrophages to the M2 phenotype, so blocking CSF1, or Conclusions depleting CSF1R-expressing cells promotes the development of antitumor M1 macrophages, and has been shown to be In a malignancy in which no major treatment advances have efficacious in animal studies (81). A relatively specific small- occurred in the past 30 years, we are now witnessing a second molecule inhibitor of CSF1R (PLX3397; Plexxikon) is now coming of immunotherapy in the form of PD-1/PD-L1 block- undergoing clinical evaluation in a phase I/II trial in combi- ade. However, modern immunotherapy is still in its infancy in nation with pembrolizumab in patients with advanced cancers UBC; in addition to immune checkpoint blockade, a number including UBC (NCT02452424; Table 1). mAbs targeting of new metabolic, vaccine, agonist, and tumor microenviron- CSF1R have also been generated (FPA008, Five Prime Thera- mental approaches are in development. Ultimately, the appli- peutics; emactuzumab, Hoffmann-La Roche). Targeting TAMs cation of these agents in UBC should be driven by a more represents a potentially important alternative approach to comprehensive understanding of the factors limiting an antitumor immunotherapy, particularly in combination with tumor immune response, both in patients and in physiologi- T-cell–directed agents like anti–PD-1/PD-L1 (Table 1). cally relevant animal models.

IDO1: a metabolic approach to T-cell dysfunction. Indoleamine 2,3- Disclosure of Potential Conflicts of Interest dioxygenase 1 (IDO1) is a cytosolic enzyme that mediates N.M. Hahn is a consultant/advisory board member for AstraZeneca/ the rate-limiting step of tryptophan metabolism (82). In the MedImmune, Bristol-Myers Squibb, Genentech/Roche, Merck, and OncoGenex tumor microenvironment, it is expressed by myeloid-derived Pharmaceuticals. C.G. Drake reports receiving commercial research grants from suppressor cells and TAMs in response to inflammation (83). Aduro Biotech, Bristol-Myers Squibb, and Janssen; has ownership interest T cells are critically dependent on tryptophan for their activity, (including patents) in Compugen, NexImmune, Potenza Therapeutics, and Tizona Therapeutics; and is a consultant/advisory board member for Agenus, and the catabolism of tryptophan by IDO1 profoundly suppresses Argos Therapeutics, Bristol-Myers Squibb, Compugen, F-star Biotechnology T-cell activity (84). The small-molecule INCB024360 (Incyte (uncompensated), Janssen, and MedImmune. No potential conflicts of interest Corp.) is a selective IDO1 inhibitor currently in phase I and II were disclosed by the other authors. trials. A number of combination trials are under way, including NCT02178722, which combines INCB024360 with pembrolizu- Authors' Contributions mab for treatment in a number of diseases including UBC. Other Conception and design: A. Ghasemzadeh, T.J. Bivalacqua, N.M. Hahn, IDO inhibitors are also in development, including indoximod C.G. Drake and GDC919 (Genentech, Inc.). Indoximod has been tested in a Development of methodology: A. Ghasemzadeh, T.J. Bivalacqua, N.M. Hahn, variety of cancers including a phase II trial in refractory metastatic C.G. Drake

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Acquisition of data (provided animals, acquired and managed patients, pro- Grant Support vided facilities, etc.): A. Ghasemzadeh, T.J. Bivalacqua, N.M. Hahn, C.G. Drake A. Ghasemzadeh was supported by the NIH under award number Analysis and interpretation of data (e.g., statistical analysis, biostatistics, T32GM007309. C.G. Drake is supported by the NIH under award number computational analysis): A. Ghasemzadeh, T.J. Bivalacqua, N.M. Hahn, R01CA127153, the Patrick C. Walsh Prostate Cancer Research Fund, the One-in- C.G. Drake Six Foundation, the Prostate Cancer Foundation, and the Melanoma Research Writing, review, and/or revision of the manuscript: A. Ghasemzadeh, Alliance. T.J. Bivalacqua, N.M. Hahn, C.G. Drake Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Ghasemzadeh, C.G. Drake Received August 31, 2015; revised November 16, 2015; accepted November Study supervision: C.G. Drake 24, 2015; published OnlineFirst December 18, 2015.

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New Strategies in Bladder Cancer: A Second Coming for Immunotherapy

Ali Ghasemzadeh, Trinity J. Bivalacqua, Noah M. Hahn, et al.

Clin Cancer Res Published OnlineFirst December 18, 2015.

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