Future Approaches in Immunotherapy - CyberLeninka

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advancing cancer immunotherapy and have inspired novel therapeutic strategies. ... advances include recombinant oncolytic viruses and adoptive transfer of .... free of progression for more than 5 years.18,19 Mono- ..... secret to supercharging their antitumor potential. ..... Hematology Am Soc Hematol Educ Program.
Future Approaches in Immunotherapy Brian Rini Advances in our understanding of the complex mechanisms of immune regulation and the interactions between tumor cells and the immune system have provided a solid foundation for advancing cancer immunotherapy and have inspired novel therapeutic strategies. Optimizing the effectiveness of immunotherapy will require targeting the antitumor immune response at multiple levels, and this may be achieved through synergistic combinations. Examples include combining two cancer vaccines to achieve a “prime and boost” effect, combining two immune checkpoint inhibitors, combining immunotherapy with targeted agents, or combining immunotherapy with low-dose chemotherapy or radiation. Immune checkpoint inhibitors, such as ipilimumab and nivolumab, will likely play an important role in the future of immunotherapy. The ability to block key pathways by which tumor cells seek to evade or suppress the immune response is critical to realizing the potential of cancer immunotherapy. Other exciting advances include recombinant oncolytic viruses and adoptive transfer of chimeric antigen receptor T cells. However, many challenges remain if durable tumor eradication with minimal toxicity is to be achieved in a broader population of cancer patients. Semin Oncol 41:S30-S40 & 2014 Elsevier Inc. All rights reserved.

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ecent advances in our understanding of the underlying mechanisms of tumor-induced immune suppression and immune checkpoints are converging to advance the field of immuno-oncology. Optimizing the effectiveness of immunotherapy will require targeting the antitumor immune response at multiple levels. Immunotherapies can be broadly characterized into three classes: (1) those that increase the frequency of tumorspecific T cells (eg, tumor vaccines, interleukin [IL]-2, and cytotoxic T-lymphocyte–associated antigen 4 [CTLA-4] inhibitors); (2) those that overcome immune suppressive mechanisms within the tumor microenvironment (eg, inhibitors of programmed cell death 1 [PD-1], indoleamine-2,3-dioxygenase [IDO], lymphocyte activation gene 3 [LAG-3], and regulatory T cell [Treg] depletion); and (3) those that trigger innate immune activation and inflammation Cleveland Clinic, Cleveland, OH. Financial support for this supplement was provided by an unrestricted educational grant from Merck & Co., Inc. Conflict of interest statement: Dr Rini has received consulting fees from and performed contracted research for Bristol-Myers Squibb, GlaxoSmithKline, Merck, and Pfizer. Additionally, he has performed contracted research for Genentech. Address correspondence to Brian Rini, MD, FACP, Solid Tumor Oncology, Cleveland Clinic Main Campus, Mail Code R35, 9500 Euclid Ave, Cleveland, OH 44195. E-mail: [email protected]. 0093-7754/ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.seminoncol.2014.09.005

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in the tumor microenvironment (eg, Toll-like receptor [TLR] agonists, type I interferons, and inhibitors of myeloid-derived suppressor cells [MDSCs]).1 Many agents are now in development within each of these distinct classes (see article by Disis in this supplement), and a range of new strategies is being investigated, most notably combinations of these agents.

COMBINATION THERAPY AS FUTURE OF IMMUNOTHERAPY One strategy to improve the effectiveness of immunotherapy is to look for synergistic combinations. This may involve combining immunotherapy with traditional cytotoxics such as chemotherapy or radiation, or combining two immunotherapeutic agents that have complementary mechanisms of action.2 The former strategy has been employed for more than a decade in the form of immunoconjugates that induce both direct and immune-mediated cytotoxicity.3 Examples include radiolabeled antiCD20 monoclonal antibodies (mAbs) such as ibritumomab tiuxetan and tositumomab for the treatment of B-cell lymphoma, and conjugates of mAbs with cytotoxic agents such as gemtuzumab ozogamicin for acute myelogenous leukemia (AML) and trastuzumab emtansine for human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Combining two or more immunotherapeutic agents

Seminars in Oncology, Vol 41, No 5, Suppl 5, October 2014, pp S30-S40

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also appears promising, but the potential for additive toxicity must be considered. To date, this approach has mainly been limited to co-administration of wellcharacterized immune adjuvants, such as IL-2 or granulocyte-macrophage colony-stimulating factor (GM-CSF), together with tumor-specific mAbs, tumor-infiltrating lymphocytes (TILs), or cancer vaccines, in an attempt to stimulate the recruitment and/or activation of immune effector cells.4 More recently, multiple examples of successful immunotherapy combinations have been evaluated in preclinical models,1 and clinical studies have begun to explore the safety and effectiveness of these new combinations. Examples include combining two vaccines to achieve a so-called “prime and boost” effect, combining two immune checkpoint inhibitors with distinct mechanisms of action, or combining an immune checkpoint inhibitor with a vaccine. Another area of active investigation is the combination of immune checkpoint inhibitors with chemotherapy and targeted therapies.

Combinations of Two or More Immunotherapies The concept behind vaccine combinations is to first prime the immune response to tumor antigens, and then boost the response with a second vaccine. This approach was recently tested in patients with metastatic pancreatic cancer, using a combination of pancreatic GVAX (Cell Genesys, Inc, San Francisco, CA) and CRS-207.5 Pancreatic GVAX is a polyvalent vaccine containing irradiated, GM-CSFsecreting, allogeneic pancreatic cancer cell lines, and is administered intradermally. CRS-207 contains liveattenuated Listeria monocytogenes, which expresses mesothelin and stimulates innate and adaptive immunity. In animal models, the combination of GVAX and CRS-207 was synergistic. In the randomized phase II study of this combination, low-dose cyclophosphamide was administered prior to GVAX to inhibit Tregs, and overall survival (OS) was significantly improved by the combination of GVAX followed by CRS-207 compared with GVAX alone. Among patients who received at least three doses of vaccine (including at least one dose of CRS-207 in the combination arm), median OS was 9.7 months with the combination versus 4.6 months with GVAX alone (hazard ratio [HR] ¼ 0.44; P ¼ .007).5 Moreover, this improvement in OS was achieved with manageable toxicity (local reactions to GVAX and transient fevers, rigors and lymphopenia after CRS-207). Further follow-up and supplemental studies are needed. Additional strategies to improve the effectiveness of therapeutic vaccines, such as combinations with Treg depletion (using either denileukin diftitox or antiCD25 mAbs), homeostatic cytokines (eg, IL-7), or

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immune checkpoint inhibitors, are being investigated.1 For example, a phase I trial in patients with advanced pancreatic cancer is currently testing the combination of the CTLA-4 inhibitor, ipilimumab, with a pancreatic tumor cell vaccine (NCT00836407). Another approach that may have great potential is the combination of two immune checkpoint inhibitors (Table 1).6–17 The success of ipilimumab for the treatment of metastatic melanoma has spurred development of other immune checkpoint inhibitors, most notably anti-PD-1 and anti–programmed cell death ligand 1 (PD-L1) mAbs. CTLA-4 inhibitors enhance early T-cell activation in lymphatic tissues and increase the frequency of tumor-specific T cells, whereas inhibition of the PD-1/PD-L1 axis modulates the T-cell effector phase to overcome T-cell anergy in the tumor microenvironment.2,18 Thus, there is a strong rationale for combining an anti-CTLA-4 mAb (eg, ipilimumab or tremelimumab) with an anti-PD-1 mAb (eg, nivolumab) or anti-PD-L1 mAb (eg, MEDI4736). One of the hallmarks of immune checkpoint inhibitors is the durability of the objective responses. CTLA-4 blockade yields durable responses in approximately 10% of patients with advanced melanoma, with some patients from the initial phase II trials of ipilimumab having remained free of progression for more than 5 years.18,19 Monoclonal antibodies targeting PD-1 and PD-L1 are much earlier in their clinical development but seem to induce a durable response in up to one-third of melanoma patients.20 The hope is that the combination could yield a durable response in a much larger proportion of patients. Studies using a B16 melanoma model have demonstrated synergy between these two classes of mAbs.21 The initial phase I study of the combination of ipilimumab and nivolumab, administered either concurrently or sequentially, demonstrated a 40% objective response rate (ORR) with concurrent administration (53% at the highest dose level tested), and a high proportion of patients had Z80% tumor reduction at 12 weeks; however, more than half of patients experienced grade 3 or grade 4 immune-related adverse events (irAEs).17 These data are very encouraging, but raise some concerns about the potential for additive toxicity. A number of clinical trials are currently ongoing to test combinations of these two agents in patients with advanced melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), colon cancer, glioblastoma, and relapsed/refractory hematologic malignancies (Table 1). Of particular interest is a three-arm, randomized, phase III trial (NCT01844505) that is comparing ipilimumab and nivolumab monotherapy with the combination of both agents as first-line therapy for advanced melanoma. The outcome is eagerly anticipated.

Drug Name

Drug Target

Nivolumab þ ipilimumab IgG4 PD-1 mAb þ IgG1 CTLA-4 mAb

Phase Ib

Population Advanced melanoma

N* 86

Primary Endpoint Safety, ORR

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Table 1. Immune Checkpoint Inhibitor Combinations Under Investigation Efficacy Results Concurrent cohort (n = 53):16 ORR: 42% (22/53; CRs 17%) Clinical activity: 65% Median DoR not reached 1-yr OS rate: 82% 2-yr OS rate: 75%

Nivolumab þ ipilimumab IgG4 PD-1 mAb þ IgG1 vs ipilimumab alone CTLA-4 mAb

II

Nivolumab þ ipilimumab IgG4 PD-1 mAb þ IgG1 CTLA-4 mAb Nivolumab þ ipilimumab IgG4 PD-1 mAb þ IgG1 vs nivolumab alone CTLA-4 mAb or ipilimumab alone Nivolumab þ ipilimumab IgG4 PD-1 mAb þ IgG1 CTLA-4 mAb

II III

I

Unresectable, untreated advanced melanoma Advanced melanoma Untreated advanced melanoma Pretreated advanced NSCLC

150

ORR

Sequential cohort (n = 33):17 ORR: 20% Clinical activity: 43% Not available

170

Safety, ORR

915

Status

Not available

Active, not recruiting NCT01927419

Not available

Not available

OS

Not available

Not available

Recruiting NCT0178393811 Active, not recruiting NCT01844505

160

ORR

ORR ¼ 22/129 (17.1%); 1-yr OS rate ¼ 42%; median OS ¼ 9.6 mo; estimated median response duration ¼ 74 wks.8

Recruiting NCT01928394

46

Safety

ORR: 22% Median DoR not reached SD: 33%7 Not available

Grade 3/4 select AEs in 6/129 pts (5%), with most common being skin (16%), gastrointestinal (12%), and pulmonary (7%). Grade 3/4 pneumonitis in 3/129 (2%)8 Grade 3/4 treatment-related AEs in 48%7 Not available

Recruiting NCT01592370

N3+I1 (n = 21):10

Active, not recruiting NCT01472081

Nivolumab þ ipilimumab IgG4 PD-1 mAb þ IgG1 CTLA-4 mAb

I

Advanced NSCLC

Nivolumab þ ipilimumab IgG4 PD-1 mAb þ IgG1 CTLA-4 mAb

I

IgG4 PD-1 mAb þ IgG1 CTLA-4 mAb

I

Relapsed or 180 refractory hematologic malignancy Metastatic renal cell 97 carcinoma

Nivolumab þ ipilimumab, sunitinib, or pazopanib

Grade 3/4 AEs

Concurrent cohort (n = 53): Active, not recruiting Grade 3/4 treatment-related NCT01024231 AEs in 53%, with most common events being ↑ lipase (13%), ↑ AST (13%).16 Sequential cohort (n = 33): Grade 3/4 treatment-related AEs in 18%, with the most common AE being ↑ lipase (6%).17

Safety and tolerability

Safety and tolerability

N3+I1 (n = 21):10 ORR: 29% DoR: 4.1þ to 22.1þ wks PFS: 4.7þ to 28.1þ wks N1+I3 (n = 23):10 ORR: 39% DoR: 6.1þ to 18.3þ wks PFS: 4.3 to 26.1 wks

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Grade 3/4 treatment-related AEs in 5 pts (24%) N1+I3 (n = 23):10 Grade 3/4 treatment-related AEs in 14 pts (61%) Most common Grade 3/4 related AEs in both groups overall: ↑ lipase (16%), ↑ ALT (11%), diarrhea (9%), colitis (5%), ↑ amylase (5%)

Recruiting NCT01454102

ORR

Not available

N+S (n = 33):6 Grade 3/4 treatment-related AEs in 73%, with most common events being ↑ ALT (18%), hypertension (15%), hyponatremia (15%) N+P (n = 20):6 Grade 3/4 treatment-related AEs in 60%, with most common events being ↑ ALT/ AST (20% each), fatigue (15%) Not available Recruiting NCT02060188

Safety, OS

Not available

Not available

Recruiting NCT0201771714

Recruiting NCT019758319 Recruiting NCT02000947

N+P (n = 20):6 ORR: 45% PFS at 24 wks: 55%

Nivolumab þ ipilimumab IgG4 PD-1 mAb þ IgG1 CTLA-4 mAb

II

Nivolumab alone or IgG4 PD-1 mAb þ IgG1 NivoluCTLA-4 mAb mab þ ipilimumab vs bevacizumab MEDI4736 þ tremelimu- IgG1ĸ PD-L1 mAb þ IgG2 mab CTLA-4 mAb MEDI4736 þ tremelimu- IgG1ĸ PD-L1 mAb þ IgG2 mab CTLA-4 mAb

IIb

MK-3475 þ IgG4 PD-1 mAb chemotherapy or IMT IpilimuIgG1 CTLA-4 mAb þ IgG3 mab þ bavituximab phosphatides serine mAb Ipilimumab þ lirilumab IgG1 CTLA-4 mAb þ IgG4 KIR mAb

I

102

ORR, safety

Not available

Not available

Ib

Advanced solid tumors Advanced NSCLC

208

MTD, safety

Not reported12

I/II

Advanced NSCLC

320

ORR

Not available

Safety data available for 3 subjects. Treatment-related AEs include a grade 2 asymptomatic ↑ amylase. No grade 3/4 DLTs12 Not available

Ib

Advanced melanoma

24

Safety and ORR

Not available

Not available

Not yet recruiting NCT01984255

Advanced melanoma, NSCLC, CRPC Advanced refractory solid tumors Advanced solid tumors Advanced cancers

125

Safety and tolerability

Not available

Not available

Recruiting NCT0175058013

150

Safety

Not available

Not available

Recruiting NCT0171473915

24

Not available

Not available

Recruiting NCT02013804

Not available

Not available

Advanced solid tumors

168

Safety and tolerability Safety and tolerability Safety and tolerability

Not available

Not available

Not yet recruiting NCT02118337 Recruiting NCT01968109

I

Nivolumab þ lirilumab

IgG4 PD-1 mAb þ IgG4 KIR mAb

I

MEDI0680

PD-1 mAb

I

MEDI0680 þ MEDI4736 PD-1 mAb þ IgG1κ PD-L1 mAb BMS-986016 alone and LAG-3 mAb þ IgG4 PD-1 with nivolumab mAb

Recurrent and 96 advanced colon cancer Recurrent 260 glioblastoma

I I

150

Future approaches in immunotherapy

N+S (n = 33):6 ORR: 52% PFS at 24 wks: 78%

Recruiting NCT02039674

n

N indicates actual enrollment for trials that have reported data and estimated enrollment for all other trials. Abbreviations: AE, adverse event; CRPC, castration-resistant prostate cancer; CTLA-4, cytotoxic T-lymphocyte antigen-4; DoR, duration of response; IgG, immunoglobulin G; IMT, immunotherapy; KIR, killer cell immunoglobulin-like receptor; LAG-3, lymphocyte activation gene 3; mAb, monoclonal antibody; MTD, maximum tolerated dose; N, nivolumab; N1þI3, nivolumab 1 mg/kg þ ipilimumab 3 mg/kg; N3þI1, nivolumab 3 mg/kg þ ipilimumab 1 mg/kg; NSCLC, non-small cell lung cancer; ORR, objective response rate; OS, overall survival; P, pazopanib; PD-1, programmed cell death 1; PD-L1, programmed cell death ligand 1; pts, patients; S, sunitinib; wk, week.

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Immune checkpoint inhibitors also may play an important role in the future treatment of lung cancer.22 Ongoing studies are investigating ipilimumab and nivolumab (both in combination with platinum-based chemotherapy) in patients with advanced lung cancer. In a phase II study in patients with NSCLC, first-line treatment with nivolumab plus chemotherapy yielded an ORR of 33%, and although grade 3/4 adverse events occurred in 49% of patients, these were largely attributed to chemotherapy.23 Based on these encouraging results, several phase III trials are underway or planned, including a trial combining ipilimumab and nivolumab in advanced NSCLC. Other anti-PD-1/PD-L1 mAbs (eg, MK-3475, MPDL3280A, and MEDI4736) are also in development, and MEDI4736 will be studied in a phase III trial as maintenance therapy for NSCLC (NCT02125461). Currently, there are several ongoing early-phase trials combining MEDI4736 with tremelimumab in NSCLC and other solid tumors, and the combination of MK-3475 with either chemotherapy or immunotherapy is being evaluated in advanced NSCLC (Table 1). In addition to PD-1 and PD-L1, several additional molecules have been shown to suppress the immune response in the tumor microenvironment, including killer cell immunoglobulin-like receptor (KIR), which is expressed on natural killer (NK) cells and downregulates NK cell cytotoxic activity,24,25 and LAG-3 (CD223), a CD4-related inhibitory receptor coexpressed with PD-1 on tolerant TILs.26,27 Antibodies capable of blocking KIR (lirilumab) or LAG-3 (BMS-986016) are currently in clinical development for the treatment of advanced solid tumors either alone or in combination with ipilimumab or nivolumab (Table 1). A key to successful application of these novel agents will be to develop biomarkers that can identify which checkpoints are most clinically relevant in any given patient.

Combinations of Immunotherapy With Targeted Agents Immune checkpoint inhibitors and other immunomodulatory therapies also are being combined actively with a variety of other treatment modalities, including targeted agents, chemotherapy, and radiotherapy (RT). It has been hypothesized that combining immunotherapy with targeted agents could be synergistic because targeted agents can promote apoptosis of tumor cells, thereby enhancing tumor antigen presentation without adversely effecting immune effector cells; they also can directly modulate the immune response and improve immune-cell function, essentially acting as immune-sensitizing agents, through a variety of mechanisms.28,29 Examples of this are shown in Table 2.29 The

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multitargeted tyrosine kinase inhibitors (TKIs) imatinib and dasatinib are prime examples of agents that can have clinically relevant immunomodulatory activity, which may contribute substantially to their antitumor activity in certain settings.29,30 Recent studies suggest that these agents suppress the activity of Tregs and enhance NK-cell activity.31–33 Imatinib blocks IDO enzyme activity, thereby suppressing Treg numbers and function, and promotes dendritic cell (DC)–NK cell crosstalk,33 whereas dasatinib appears to differentially inhibit Treg function by blocking lymphocyte-specific protein tyrosine kinase (Lck), a critical kinase involved in T-cell receptor signaling.32 These findings have led to preclinical studies investigating the combination of imatinib with an anti-CTLA-4 mAb and dasatinib with an agonist anti-OX40 mAb in an effort to further stimulate the antitumor immune response. These combinations appear to have synergistic antitumor activity in animal models.30 A similar strategy currently in phase I testing in melanoma is the combination of ipilimumab with vemurafenib, which inhibits BRAFV600E. Vemurafenib has been shown to upregulate expression of tumor-associated antigens (eg, gp100 and MART1) on melanoma cells,34 suppress secretion of inhibitory cytokines (eg, IL-10) by tumor cells, and cause tumor antigen release.29,35 The hope is that adding ipilimumab will enhance activation and proliferation of T cells primed by those antigens. However, preliminary results from the phase I trial demonstrated severe hepatic toxicity with this combination,36 and the study was halted. The combination of immunotherapy with antiangiogenic agents (eg, bevacizumab, sunitinib, and pazopanib) is also supported by a strong biologic rationale. Bevacizumab has been shown to increase maturation of DCs and antigen presentation, and sunitinib can decrease the number of MDSCs and Tregs in the tumor microenvironment (Table 2).29 Consequently, the combination of bevacizumab plus ipilimumab is being evaluated in a phase I study in patients with advanced melanoma (NCT00790010), and nivolumab is being combined with sunitinib or pazopanib in patients with metastatic RCC in the CheckMate 016 phase I trial (NCT01472081). Finally, antibodies against HER2 (eg, trastuzumab) or epidermal growth factor receptor (EGFR), such as cetuximab, form immune complexes with tumor cells and enhance tumor antigen presentation and expression of co-stimulatory molecules (eg, CD40, CD80, and CD86) on DCs.37,38 Similarly, JAK2 inhibitors can enhance antigen presentation by DCs by inhibiting immunosuppressive STAT3 signaling.39,40 As a result, these agents may synergize with therapeutic vaccines. Two phase II trials are currently exploring this approach, one with trastuzumab in HER2-positive breast cancer (NCT01570036),

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Table 2. Immunomodulatory Effects of Targeted Agents and Experimental Combinations with Immunotherapy28 Agent Imatinib

Primary Target(s) Multitargeted (BCR/ABL, PDGF, KIT, etc.)

Effect on Immune System

Experimental Combinations

 Inhibits IDO  Decreases number and activity of Treg  Promotes DC-NK cell crosstalk

Preclinical with anti-CTLA-4 mAb in GIST Phase II with BCR-ABL vaccine in CML Phase III with IFN-α in CML

Dasatinib

Multitargeted (BCR/ABL, Src family kinases)

 Inhibits Treg function by blocking Lck

Preclinical with an agonist anti-OX40 mAb

Vemurafenib

BRAFV600E

 Increases expression of tumor-associated

Phase I with ipilimumab (NCT01400451)

antigens pg100, MART1, etc

 Decreases tumor secretion of immunosuppressive cytokines (eg, IL-10) Bevacizumab

VEGF

 Increases DC maturation, antigen presentation, and T-cell activation

 Shifts DC maturation away from MDSC

Preclinical with adoptive T-cell transfer Phase I with ipilimumab (NCT00790010) Phase III with IFN-α in metastatic RCC

phenotype Sunitinib/ Pazopanib

VEGFR

 Inhibits STAT3 signaling  Decreases numbers and activity of MDSCs and Treg cells

Trastuzumab

HER2

Preclinical with adoptive T-cell transfer in HCC and RCC models Preclinical with anti-CD137 agonist mAb plus IL-12 in colon cancer model

 Primes tumor-specific CTLs  Boosts NK cell secretion of IFN-γ and ADCC

Preclinical with 4-1BB agonist mAb Preclinical with anti-PD-1 mAb Phase I with IL-12 and paclitaxel in HER2þ breast cancer Phase II with HER2 peptide vaccine

Cetuximab

EGFR

 Mediates complement fixation and ADCC  Increases MHC class I & II expression  Increases DC priming of tumor-specific CTLs

Phase II with EGFR vaccine (NCT00305760)

JAK2 inhibitors

JAK2, STAT3

 Increases DC maturation, antigen presentation,

Preclinical with DC vaccines

and T-cell activation

 Inhibits STAT3 signaling  Decreases PD-L1 expression on tumor cells Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; CML, chronic myelogenous leukemia; CTL, cytotoxic T lymphocyte; CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; DC, dendritic cell; EGFR, epidermal growth factor receptor; GIST, gastrointestinal stromal tumor; HCC, hepatocellular carcinoma; HER2, human epidermal growth factor receptor 2; IDO, indoleamine-2,3-dioxygenase; IFN, interferon; IL, interleukin; mAb, monoclonal antibody; MHC, major histocompatibility complex; NK, natural killer; PD-1, programmed cell death 1; PDGF, platelet-derived growth factor; PD-L1, programmed cell death ligand 1; RCC, renal cell carcinoma; Treg, regulatory T cell; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

and the other with cetuximab in pancreatic cancer (NCT00305760). Most other combinations of immunotherapy with targeted agents are still in preclinical testing.

Combinations of Immunotherapy With Radiotherapy or Chemotherapy Radiation-induced damage to tumor cells can facilitate release of tumor-associated antigens and high mobility group box 1 (HMGB1) protein, which binds to TLR4 and serves as a “danger signal” that attracts immune effector cells to the tumor

microenvironment.2,41,42 Radiation also makes tumor cells more susceptible to immune-mediated killing, in part via increased expression of major histocompatibility complex (MHC) class I and death receptors (eg, FAS).41 In fact, the available evidence suggests that an intact immune system may be critical for RT to exert its full antitumor effect.43 Early studies showed that combining RT with a therapeutic vaccine resulted in tumor-specific immune responses in the majority of patients.44,45 More recently, studies in a mouse model demonstrated that an anti-PD-L1 mAb enhanced the efficacy of RT through a cytotoxic T lymphocyte (CTL)-

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dependent mechanism, and the combination synergistically reduced the number of tumor-infiltrating MDSCs.46 Currently, a randomized phase II study in patients with metastatic melanoma is comparing RT plus ipilimumab with ipilimumab monotherapy (NCT01689974). A critical issue that must be addressed with this strategy is the optimal timing of RT relative to administration of immunotherapy.43 The combination of immunotherapy with chemotherapy has long been considered to be antagonistic because chemotherapy is often myelosuppressive and could potentially deplete immune effector cells. However, mounting evidence suggests that some chemotherapy agents, such as cyclophosphamide (depending on the dose and timing of administration), may improve antitumor immunity through a variety of mechanisms including depletion of Tregs, enhanced activation and proliferation of T cells and B cells, infiltration of tumor-specific lymphocytes into the tumor microenvironment, and activation of myeloid DCs that secrete more IL-12 and less IL-10.2,47–50 Early studies demonstrated that lowdoses of cyclophosphamide could augment the immune response to therapeutic vaccines,51,52 and an animal model demonstrated strong additive effects in combination with an allogeneic tumorcell vaccine engineered to secrete GM-CSF.53 This strategy was recently tested with a murine version of prostate GVAX. Administration of low-dose cyclophosphamide 1 day prior to the vaccine appeared to overcome T-cell tolerance and resulted in proliferation of tumor-specific CTLs.54 In this model, the dose and timing of cyclophosphamide was critical. Immunomodulation was only achieved with doses of cyclophosphamide that did not deplete T cells. This is now being tested clinically with breast GVAX, and early results suggest that a cyclophosphamide dose of approximately 200 mg/m2 enhances the immune response, whereas higher doses are immunosuppressive.2

NOVEL IMMUNOTHERAPY STRATEGIES Some of the newest approaches to immunotherapy are adoptive transfer of engineered T cells and oncolytic viruses. These are still in very early phases of development but appear to hold great promise.55 Similar to the way a patient’s autologous DCs have been manipulated to express tumor antigens or secrete pro-inflammatory cytokines, autologous T cells are being genetically engineered to express chimeric antigen receptors (CARs) that typically consist of an mAb fragment fused to the T-cell receptor signaling domain.56 The antibody portion of the molecule can recognize a tumor antigen without the need for self human leukocyte antigen (HLA), and that induces a signal to activate the T cell.

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This approach has many advantages, most importantly HLA-independent antigen recognition. Early results were disappointing, but second-generation chimeric antigen receptor T (CAR-T) cells that incorporated co-stimulatory signaling domains (eg, CD28, 4-1BB, or OX-40) have proven to be effective.57,58 CAR-T cells have been investigated recently in several pilot studies in lymphoma.56,59,60 In two studies conducted in patients with chronic and acute lymphocytic leukemia who received conditioning chemotherapy (to deplete Tregs) followed by autologous CAR-T cells engineered to recognize CD19, the transferred CAR-T cells expanded more than 1,000-fold in vivo and persisted for more than 6 months.61,62 More importantly several patients achieved durable complete or partial responses. However, adoptive transfer of CAR-T cells can also cause severe toxicity due to cytokine release syndrome. Both autologous and allogeneic anti-CD19 CAR-T cells are now being investigated in more than a dozen clinical trials.56 Use of oncolytic viruses as anticancer agents is an area of active clinical research. An oncolytic virus can selectively infect, replicate in, and kill tumor cells, either naturally or through genetic engineering, while causing limited or no damage to normal cells.4,63 Targeting of the virus to tumor cells and/or selective replication in tumor cells is typically engineered into recombinant viruses. Clinical trials using a recombinant oncolytic virus were conducted approximately 20 years ago.64,65 Since then, several viruses exhibiting oncolytic activity have been identified and studied to determine their potential as anticancer agents.4,55 Examples include herpes simplex virus (HSV), adenovirus, pox virus, and measles virus, which naturally infect humans, as well as several animal viruses (eg, vesicular stomatitis virus, Newcastle disease virus, and myxoma virus) that can be genetically modified to infect human cells. The first oncolytic virus was approved in China in 2005 for treating nasopharyngeal cancer (in combination with chemotherapy)66, and several are being tested in phase III clinical trials in the United States.67 Infection and lysis of tumor cells by an oncolytic virus can stimulate a robust antitumor immune response through a variety of mechanisms, including release of tumor antigens, proinflammatory cytokines, and danger signals such as HMGB1.4,63 Oncolytic viruses also can be engineered to direct the infected tumor cells to produce pro-inflammatory cytokines such as GM-CSF. For example, talimogene laherparevec (T-VEC) is a recombinant oncolytic HSV that produces GM-CSF and has demonstrated good clinical activity after injection directly into metastatic melanoma lesions. In a phase II trial, it produced a 26% response rate against systemic disease, following local injection into accessible

Future approaches in immunotherapy

tumors.68 A pivotal phase III trial (OPTiM) of T-VEC has been completed in melanoma.69 In 436 patients with unresectable stage IIIB-IV melanoma, T-VEC yielded a 26.4% ORR (10.8% complete response [CR]) compared with 5.7% ORR (0.7% CR) in patients treated with GM-CSF alone.69 Pexastimogene devacirepvec (Pexa-Vec) is an oncolytic poxvirus that also produces GM-CSF. It has been tested in several phase I/II clinical trials and has demonstrated promising activity in patients with hepatocellular carcinoma.70–75

FORWARD LOOK AT THE ROLE OF IMMUNOTHERAPY Over the next 5 to 10 years, the focus of clinical research will be to identify and assess new combinations with synergistic antitumor activity and reduced toxicity. If the ultimate goal of cancer immunotherapy is to achieve durable tumor eradication with minimal toxicity, many obstacles and challenges lie ahead before that promise can be realized. Identification of appropriate tumor antigens for development of targeted immunotherapies is a major challenge. The ideal tumor antigen is truly tumor specific and essential for tumor cell growth and survival, but this is often different for each individual tumor.55 Most mAbs and CARs target tumor-associated cell surface antigens, but this can lead to destruction of normal cells (eg, CD20 or CD19-positive B cells) and selection of tumor variants (through immunoediting) that no longer express that antigen. To reduce the effect of immunoediting, the tumor antigen being targeted must be essential for tumor survival. As effective immunotherapies become more widely available and integrated into routine clinical practice outside of a few specialized cancer centers, it also will be important to identify biomarkers to predict response to immunotherapy and identify those patients who are most likely to benefit. For example, selection of PD-L1positive tumors for treatment with a PD-1 or PD-L1 inhibitor appears to be a rational strategy, but a reliable predictive biomarker for response to ipilimumab has been elusive.18 Another challenge is maximizing the number of patients who achieve a durable benefit from immunotherapy. The hope is that combining CTLA-4 inhibitors with PD-1/PD-L1 inhibitors may move us closer to achieving that goal, and combinations of immune checkpoint inhibitors with targeted agents (eg, inhibitors of the BRAF/MEK pathway in melanoma) could provide even greater benefit, but at what cost in terms of toxicity? A final challenge may be “dialing in” the amount of checkpoint inhibition needed for a particular patient and a given time point in disease to optimize the balance between efficacy and toxicity.

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The early success of adoptive T-cell therapy for melanoma and leukemia provides a glimpse of what immunotherapy is capable of, but the obstacles are overcoming tumor-induced immune suppression and making this technology available to a broader patient population. Perhaps CAR-T cells are the answer, and early evidence suggests that combining them with co-stimulatory molecules, mitogenic cytokines, or immune checkpoint inhibitors may be the secret to supercharging their antitumor potential. Therapeutic vaccines for advanced cancer also have new life, and new combinations of cancer vaccines with a variety of immunomodulatory agents are actively being investigated. However, successful development of novel vaccine strategies and integration of those strategies into routine clinical practice still face many challenges related to identification of appropriate tumor antigens, overcoming immune suppression, predictive biomarkers, and patient selection.76,77

CONCLUSIONS Advances in our understanding of the mechanisms of immune regulation and the complex interactions between tumor cells and the immune system have provided a solid foundation for the development of combinatorial approaches and have inspired novel immunotherapy strategies. However, much work remains to be done to refine those strategies and integrate them into routine clinical practice. The recent success of immune checkpoint inhibitors and therapeutic vaccines has sparked a renaissance in immunotherapy, and these agents are now being combined with a wide array of other immunotherapies and with targeted agents, radiotherapy, and chemotherapy in the hope of achieving synergistic antitumor activity. The key to the long-term success of cancer immunotherapy is to identify optimal tumor antigens/targets, develop predictive biomarkers, and overcome issues with toxicity.

Acknowledgments The author would like to thank Marithea Goberville, PhD, and Chelsey Goins, PhD, for their assistance in drafting the manuscript, and Heather Tomlinson, ELS, for her assistance with preparing the manuscript for submission.

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