Immuno-Oncology Combinations: a Review of Clinical Experience and Future Prospects

Published OnlineFirst October 23, 2014; DOI: 10.1158/1078-0432.CCR-14-1457

Clinical Cancer Review Research

Immuno-oncology Combinations: A Review of Clinical Experience and Future Prospects

Scott J. Antonia1, James Larkin2, and Paolo A. Ascierto3

Abstract Immuno-oncology is an evolving treatment modality that includes immunotherapies designed to harness the patient’s own immune system. This approach is being studied for its potential to improve long-term survival across multiple tumor types. It is now important to determine how immunotherapies may be most effectively used to achieve the best possible patient outcomes. Combining or sequencing immunotherapies that target distinct immune pathways is a logical approach, with the potential to further enhance the magnitude of the antitumor immune response over single agents. Early clinical data in patients with melanoma treated with two immune checkpoint inhibitors, ipilimumab and nivolumab, suggest support for this combination approach. Numerous other combination approaches are being evaluated in early-phase clinical trials; however, their clinical activity remains unknown. Clinical experience to date has shown that when combining an immuno-oncology agent with an existing therapeutic modality, it is important to determine the optimal dose, schedule, and sequence. Clin Cancer Res; 20(24); 1–11. 2014 AACR.

Introduction and have been associated with significant toxicity and limited Tumors avoid immune destruction by a range of complex efficacy; these factors restrict use to healthy patients and only and often overlapping mechanisms that disrupt key compo- a select group of these patients will derive benefit (6, 7). nents of the immune system involved in mounting an Immuno-oncology is an evolving treatment modality effective antitumor response (1–4). Tumors can avoid rec- that includes immunotherapies designed to target and ognition and elimination by the immune system by disrupt- harness the patient’s immune system directly to kill tumor ing antigen presentation mechanisms, either through down- cells (8, 9). Numerous strategies for overcoming tumor regulation of MHC class I molecules or by disabling antigen- immune evasion are under evaluation (Table 1). Because processing machinery. Alternatively, or additionally, tumors these approaches directly target the patient’s immune sys- may suppress the immune system by disrupting pathways tem, they have the potential for activity across multiple involved in controlling T-cell inhibition (checkpoint) and types of cancer. Examples of immunotherapeutic appro- activation (3, 5), or by recruiting immunosuppressive cell aches under clinical investigation include T-cell checkpoint types, such as regulatory T cells (Treg) and myeloid-derived inhibitors or agonists for T-cell–activating pathways, novel suppressor cells (MDSC). The release of factors, including cytokines such as IL12 and IL15, therapeutic vaccines, adenosine and prostaglandin E2, and the enzyme indolea- elimination of immunosuppressive cells, and other agents mine 2,3-dioxygenase (IDO) is another mechanism that and approaches designed to enhance immune cell function tumors may use to suppress immune activity (3). (Table 1; refs. 10–12). The idea of targeting the immune system as a therapeutic Since the approval of IL2, sipuleucel-T (a therapeutic approach in cancer is not new. Cytokines [interleukin-2 (IL2) vaccine composed of recombinant antigen protein designed and interferon-a (IFNa)] have been used for decades, pre- to stimulate T-cell responses) and ipilimumab [a cytotoxic dominantly in patients with renal cell carcinoma (RCC) and T-lymphocyte antigen 4 (CTLA-4) immune checkpoint melanoma. However, these cytokines are not target specific, inhibitor] were the first immunotherapies to be approved for patients with cancer. Sipuleucel-T was approved in 2010 for asymptomatic or minimally symptomatic metastatic

1Moffitt Cancer Center and Research Institute, Tampa, Florida. 2The Royal castrate–resistant prostate cancer (CRPC) and ipilimumab Marsden, London, United Kingdom. 3Istituto Nazionale Tumori Fondazione in 2011 for unresectable or metastatic melanoma. Both G. Pascale, Naples, Italy. agents were shown to significantly improve overall survival Note: S.J. Antonia and P.A. Ascierto share senior authorship. (OS) in phase III clinical trials (Fig. 1; refs. 13–16). Corresponding Author: Scott J. Antonia, Moffitt Cancer Center and Monoclonal antibodies targeting programmed death-1 Research Institute, Tampa, FL 33612. Phone: 813-745-8470; E-mail: (PD-1) ligand (PD-L1) interaction, another immune check- Scott.Antonia@moffitt.org point pathway, are the most advanced in clinical develop- doi: 10.1158/1078-0432.CCR-14-1457 ment after ipilimumab and sipuleucel-T, and various agents 2014 American Association for Cancer Research. are being tested in clinical trials across a range of tumor types.

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Table 1. Potential strategies for overcoming tumor immune evasion mechanisms and examples of agents in clinical development (12)

Treatment strategy Examples of agents in clinical development Reversing the inhibition of adaptive immunity (blocking T-cell checkpoint pathways)

* Inhibiting the CTLA-4 checkpoint Ipilimumab: approved for melanoma moleculea Tremelimumab: phase II for malignant mesothelioma, HCC, melanoma

* Inhibiting the interaction between PD-1 Nivolumab (anti–PD-1): phase III for melanoma, NSCLC, RCC checkpoint and its ligandsa Pembrolizumab (MK-3475; anti–PD-1): phase III for NSCLC, melanoma MPDL3280A (RG7446; anti–PD-L1): phase III for NSCLC Pidilizumab (CT-011; anti–PD-1): phase II for FL, prostate, pancreatic, melanoma AMP-514 (MEDI0680; anti–PD-1): phase I for solid tumors MEDI4736 (anti–PD-L1): phase I for solid tumors AMP-224 (recombinant PD-L-Fc fusion protein): phase I for solid tumors rHIgM12B7 (anti–PD-L2): phase I for melanoma

* Inhibiting the LAG-3 checkpoint IMP321: phase I for breast, RCC; phase II for melanoma molecule BMS-986016: phase I for solid tumors

* Inhibiting the TIM-3 checkpoint No agent undergoing clinical evaluation

* Inhibiting the adenosine A2A receptor No agent undergoing clinical evaluation

Switching on adaptive immunity (promoting T-cell costimulatory receptor signaling using agonist antibodies) * Promoting CD137 signaling Urelumab: phase I for B-cell NHL, CLL, solid tumors PF-05082566: phase I for NHL

* Enhancing OX-40 signaling MEDI6469: phase II for breast, prostate, solid tumors

* Promoting GITR signaling TRX518: phase I for melanoma, solid tumors

* Enhancing CD27 signaling CDX-1127: phase I for CD27-expressing hematologic malignancies and solid tumors * CD40 Activation CP-870,893: phase I for pancreatic, melanoma Chi Lob 7/4: phase I for advanced malignancies * Systemic recombinant IL21 Denenicokin: phase II for melanoma, ovarian and phase I for RCC, NHL administration (range of effects that enhance immune cell function) * Systemic recombinant IL15 rhIL 15: phase I for melanoma, kidney, NSCLC, SCHN administration (range of effects that enhance immune cell function) * Systemic recombinant IL7 rhIL 7: phase II for various solid tumors administration (range of effects, including on T-cell development)

Improving the function of innate immune cells * Manipulating the activation of NK-cell Lirilumab: phase II for AML (maintenance); phase I for solid tumors inhibitory receptors (KIR) * Stimulating macrophages and DCs Toll-like receptor agonists: Bacillus Calmette-Guerin (TLR 2/4 agonist): approved for bladder carcinoma Hiltonol (TLR7 agonist): phase II various solid and hematologic malignancies Imiquimod (TLR7 agonist): approved basal cell carcinoma Resiquimod (TRL7/8 agonist): phase I/II various solid and hematologic malignancies CpG 7909 (TLR 9 agonist): phase II various solid tumors

Activating the immune system (potentiating immune-cell effector function) * IDO inhibition INCB024360: phase II for melanoma, EOC, PPC, FTC Indoximod: phase II for breast, prostate (Continued on the following page)

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Table 1. Potential strategies for overcoming tumor immune evasion mechanisms and examples of agents in clinical development (12) (Cont'd )

Treatment strategy Examples of agents in clinical development

* Inhibition of TGF-b signaling GC1008; phase I for melanoma, RCC LY2157299: phase I/II various solid tumors TEW 7197: phase I solid tumors IMC-TR1 (LY3022859): phase I solid tumors a * Systemic IL2 or IFNa administration Agents approved a * Various vaccine-based strategies Various approaches under clinical evaluation

Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; EOC, epithelial ovarian cancer; FL, follicular lymphoma; FTC, fallopian tube cancer; GITR, glucocorticoid-induced tumor necrosis factor related gene; HCC, hepatocellular carcinoma; NHL, non-Hodgkin lymphoma; PPC, primary peritoneal cancer; SCHN, squamous cell head and neck cancer; TIM, T-cell immunoglobulin mucin; TLR, Toll-like receptor. aApproaches in which approved compounds or investigational compounds are being studied in phase III trials.

One of the more exciting aspects of immunotherapies is Although targeting the immune system has emerged as an demonstrated with data from clinical trials for ipilimu- effective treatment approach for patients with CRPC and mab, nivolumab, and pembrolizumab that show the metastatic melanoma (17, 18), for the development of this potential for long-term survival. In a phase III study of treatment modality to progress, it is important to determine ipilimumab in previously treated patients with metastatic how agents should be used to achieve the best possible melanoma (study MDX010-020), the survival rate at 2 patient outcomes. Combining immunotherapies with other and 3 years was 25% for each (17). In addition, in a established and investigational cancer therapies is a ﬁeld of pooled analysis of data from 12 ipilimumab clinical active investigation, with a multitude of approaches under studies with follow-up of up to 10 years in some patients, consideration. This review focuses on (i) combining or an OS plateau started at approximately 3 years and the 3- sequencing immunotherapies that target distinct immune year survival rate was 22% (18). The PD-1 immune pathways, particularly T-cell checkpoints, and (ii) combin- checkpoint inhibitors nivolumab and pembrolizumab ing immunotherapies with existing therapeutic modalities, have also shown durable responses in phase I studies speciﬁcally BRAF-targeted therapies, chemotherapies, and (19–21). radiotherapy.

1.0 0.9 n = 17 0.8

Figure 1. Targeting two distinct 0.7 n = 53 immune checkpoint pathways: 0.6 interim data from a phase I study of 1-year survival concurrent ipilimumab and 0.5 of 82% nivolumab. Patients with advanced 0.4 95% CI melanoma treated with ipilimumab in combination with nivolumab had 0.3 (69.0%–94.4%) a preliminary 1-year OS rate of

Proportion of survival 0.2 82%. These data provided the Censored Died/treated rationale for initiation of a phase III 0.1 1 mg/kg nivolumab + 3 mg/kg ipilimumab 2/17 trial of the ipilimumab/nivolumab All concurrent regimen 9/53 combination in previously 0.0 untreated patients with metastatic 03691215 18 21 24 27 30 33 36 melanoma. Reprinted with Months permission from Wolchok et al. (16). Patients at risk 1 mg + 3 mg 17 16 16 14 10 5 3 2 2 1 0 0 0 All concurrent 53 47 36 29 19 10 7 4 4 3 1 1 0

CCR Reviews

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Combining immunotherapies that target distinct colon adenocarcinoma tumors that were largely resistant to immune pathways single antibody treatment (22). Combining or sequencing immunotherapies that target distinct immune pathways is a rational strategy to T-cell checkpoint inhibition combined with agonistic determine whether the magnitude of the antitumor antibodies against T-cell costimulatory receptors immune response may be improved over that generated In theory, if agents designed to release the checkpoint- with a single agent. Potential combination approaches mediated inhibition of T cells were combined with agonist undergoing clinical evaluation include dual T-cell check- antibodies designed to enhance costimulatory T-cell signal- point inhibition, T-cell checkpoint inhibition combined ing, a more effective immune response may be generated with immunomodulatory antibodies designed to enhance (5). To date, no data are available from clinical trials T-cell activity through agonistic interaction with costimu- evaluating these combinations, but studies are in progress. latory receptors (aiming to switch on adaptive immunity), CD40 plays a key role in the development of T-cell–depen- T-cell checkpoint inhibition combined with approaches to dent antitumor immunity, and is essential in enabling improve the function of innate immune cells, and T-cell antigen-presenting cells to process and present antigen checkpoint inhibition combined with other approaches to effectively to T cells (26–28). Combining T-cell checkpoint enhance the immune response (Table 2; refs. 3, 5, 12). blockade (using anti–CTLA-4 agent tremelimumab) with an agent that targets the costimulatory molecule CD40 Dual T-cell checkpoint inhibition (CP-870,893) is being investigated in a phase I trial in Given that T-cell checkpoint inhibitors (e.g., ipilimu- patients with melanoma (12). mab, nivolumab, pembrolizumab) have shown single- Other agonist antibodies designed to target receptors, agent clinical activity in several tumor types (5, 13), including OX-40, CD27, GITR, and CD137, are in devel- and preclinical data suggest checkpoint molecules may opment. The clinical evaluation of these agents as mono- act synergistically to regulate T-cell function and pro- therapy is at an early stage, although the limited data mote tumor immune escape, it is rational to evaluate available suggest they can be safety administered to patients. whether combining checkpoint inhibitors improves Data from a large phase I trial with urelumab (anti-CD137) activity, achieving an OS benefit in a greater proportion in more than 100 patients did show liver toxicity, with of patients compared with either agent alone (Fig. 2; 2 deaths reported at higher doses. Clinical evaluation of refs. (16, 22–24). Initial support for dual T-cell check- urelumab is continuing at lower doses in advanced solid point inhibition has come from a phase I study in tumors and hematologic malignancies (29). Evaluating which patients with advanced stage III or IV melanoma combinations of these antibodies with checkpoint inhibi- were treated with both ipilimumab (1 or 3 mg/kg) and tors and other immunotherapies is an exciting possibility, nivolumab (0.3 mg/kg, 1 mg/kg, or 3 mg/kg) in a but one that should be evaluated with caution. concurrent or sequenced regimen (16, 25). An objective Another agonistic therapeutic approach that was evalu- response rate (ORR) rate of 40% was achieved in ated with catastrophic effects was the CD28 agonist patients treated with the concurrent regimen (ORR was TGN1412. In a first-in-human phase I trial, TGN1412 53% at the maximum tolerated dose, nivolumab 1 mg/ administration resulted in a cytokine storm that caused kg, ipilimumab 3 mg/kg). The preliminary 1-year OS severe adverse events in the six volunteers (30). As explained rate with the concurrent regimen was 82% [95% con- by Curran and colleagues (31), CD28 is widely expressed on fidence interval (CI) 69.0–94.4; Fig. 1; ref. 16]. These all mature T-cell populations; therefore, an agonistic CD28 promising results prompted the initiation of a phase III antibody may be expected to have a polyclonal "super study (CheckMate 067) to further evaluate concurrent agonist" effect—this is in contrast to other costimulatory treatment with ipilimumab and nivolumab (12). modules, such as CD137 or OX-40, which are only Phase I studies are in progress to evaluate ipilimumab expressed on a proportion of T cells, so agonist antibodies plus nivolumab in patients with a range of solid tumors are likely to have a more selective effect. [including RCC, non–small cell lung cancer (NSCLC), colon cancer, triple-negative breast cancer, gastric cancer, T-cell checkpoint inhibition combined with pancreatic cancer, and small-cell lung cancer (SCLC); ipi- approaches to improve the function of innate immune limumab plus pembrolizumab (anti–PD-1) in patients cells with melanoma, RCC, and NSCLC; tremelimumab (an Adaptive immune responses to cancer involve various anti–CTLA-4 agent) plus MEDI 4736 (anti–PD-L1 agent) components of innate immunity. In view of this, combining in NSCLC; and an anti-lymphocyte activation gene 3 (LAG- therapies designed to enhance T-cell function with agents 3) monoclonal antibody BMS-986016 plusnivolumab designed to improve innate immune cell function are (anti–PD-1) in patients with solid tumors (Table 2; worthy of evaluation. Natural killer (NK) cells are innate ref. 12)]. The latter combination is supported by preclinical effector cells that maintain tolerance to self-tissue via the data that showed strong synergistic antitumor activity when expression of killer cell immunoglobulin-like receptors both the PD-1 and LAG-3 immune checkpoint pathways (KIR), which negatively regulate NK-cell activity by binding were blocked (23). Dual anti–LAG-3/anti–PD-1 antibody to the MHC class I molecules expressed on most "normal" treatment cured most mice of established fibrosarcoma and cells (32–35). Tumor cells may appear like normal cells by

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Table 2. Combination approaches in clinical development (12)

Selected clinical trials of immunotherapies

Drugs Target Development phase/tumor type Dual T-cell checkpoint blockade Ipilimumab þ nivolumab CTLA-4 þ PD-1 Phase III: melanoma; phase I/II: RCC, colon, NSCLC, triple-negative breast cancer, gastric cancer pancreatic cancer, and SCLC Ipilimumab þ pembrolizumab CTLA-4 þ PD-1 Phase I: melanoma, RCC, and NSCLC Tremelimumab þ MEDI4736 CTLA-4 þ PD-L1 Phase I: NSCLC, solid tumors Nivolumab þ BMS-986016 PD-1 þ LAG-3 Phase I: solid tumors T-cell blockade þ costimulatory receptor agonists CP-870,893 þ tremelimumab CTLA -4 þ CD40 Phase I: metastatic melanoma T-cell blockade þ improving the function of innate immune cells Lirilumab þ ipilimumab CTLA-4 þ KIR Phase I: solid tumors Lirilumab þ nivolumab PD-1 þ KIR Phase I: solid tumors T-cell blockade þ other immune system activators Denenicokin þ ipilimumab CTLA-4 þ IL21 Phase I: melanoma Denenicokin þ nivolumab PD-1 þ IL21 Phase I: solid tumors INCB024360 þ ipilimumab CTLA-4 þ IDO Phase I: melanoma Indoximod þ sipuleucel-T IDO þ vaccine Phase II: prostate Nivolumab þ gp100, NY-ESO-1 PD-1 þ vaccine Phase I: melanoma Ipilimumab þ sipuleucel-T CTLA-4 þ vaccine Phase II: prostate cancer Ipilimumab þ TriMix-DC CTLA-4 þ vaccine Phase II: melanoma Ipilimumab þ NY-ESO-1 vaccine CTLA-4 þ vaccine Phase I: melanoma Ipilimumab þ adoptive cell transfer CTLA-4 þ passive Phase I/II: melanoma immunotherapy Selected clinical trials of immunotherapies (excluding ipilimumab) in combination with other treatment modalities

Drugs Target Combination treatment Development phase/tumor modality type

Tremelimumab CTLA-4 Geﬁtinib Phase I: NSCLC Nivolumab PD-1 Chemotherapya Phase I: NSCLC Dasatinib Phase I: CML Bevacizumab Phase I: NSCLC Erlotinib Phase I: NSCLC Sunitinib or pazopanib Phase II: RCC Pembrolizumab PD-1 Pazopanib Phase I: RCC Lenalidomide þ Phase I: multiple myeloma dexamethasone MPDL3280A PD-L1 Bevcizumab Phase II: RCC Erlotinib Phase I: NSCLC Vemurafenib Phase I: melanoma MEDI14736 PD-L1 Trametinib dabrafenib Phase I/II: melanoma Pidilizumab PD-1 Rituximab Phase II: FL Gemcitabine Phase II: pancreatic FOLFOX Phase II: CRC (completed) Sipuleucil-T þ Phase II: prostate cancer cyclophosphamide IMP321 LAG-3 Paclitaxel Phase I: breast (completed) Urelumab CD137 Rituximab Phase I: NHL Chemotherapy Phase I: solid tumors PF-05082566 CD137 Rituximab Phase I: NHL CP-870,893 CD40 Paclitaxel/carboplatin Phase I: solid tumors (completed) Denenicokin IL21 Sunitinib Phase I/II: RCC Sorafenib Phase I/II: RCC Rituximab Phase I: NHL Indoximod IDO Docetaxel Phase II: breast

Abbreviations: CML, chronic myeloid leukemia; CRC, colorectal cancer; FL, follicular lymphoma; NHL, non-Hodgkin lymphoma. aChemotherapy regimens include gemcitabine/cisplatin, pemetrexed/cisplatin, and carboplatin/paclitaxel.

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tive immune cells. Data from preclinical studies suggest that 100 inhibiting IDO can promote the proliferation, survival, and function of various immune cells [e.g., T cells, NK cells, and 80 dendritic cells (DC)], reduce the generation of Tregs, and signiﬁcantly inhibit tumor growth (42, 43). Furthermore, Combination studies in murine models showed that host-derived IDO 60 can suppress the antitumor activity of an anti–CTLA-4 PD-1 pathway blockade antibody. However, inhibition or absence of IDO com- 40 bined with therapies targeting immune checkpoints, such

Percentage alive Percentage as CTLA-4, PD-1/PD-L1, and GITR, acts synergistically to CTLA-4 pathway blockade control tumor growth and improve OS (44). Thus, com- 20 bining an agent that inhibits IDO with another immunotherapy would appear to be a rational approach and is being 0 evaluated in several clinical trials (Table 2). A phase II trial is 01234evaluating the IDO inhibitor indoximod in combination Years with the therapeutic vaccine sipuleucel-T in patients with prostate cancer (Table 2; ref. 12). CCR Reviews Adoptive cell transfer and T-cell engineering. Adoptive cell transfer (ACT) involves the collection of tumor-infil- Figure 2. Hypothetical effect on OS of blocking two T-cell checkpoint trating lymphocytes (TIL) from patients, the in vitro expan- pathways. Adapted with permission from Urba (24). sion of autologous lymphocytes with reactivity to tumor antigens, and the subsequent transfer back to the patient, retaining or upregulating MHC class I to escape immuno- with the expectation that the tumor-specific lymphocytes surveillance by NK cells (33). Lirilumab is an anti-KIR will attack the tumor (11, 45). ACT has demonstrated antibody that blocks the inhibitory KIR signal, thereby durable complete responses in patients with melanoma potentiating NK-cell killing of tumor cells, despite expres- (45). In a phase II study, 20 of 93 patients with metastatic sion of MHC I. A regimen designed to enhance innate and melanoma (22%) had durable, complete remissions adaptive immunity, respectively, could theoretically (>3–7 years) after treatment with IL2 and ACT of TILs. achieve more favorable biologic and clinical activity com- In addition to the expansion and transfer of TILs, pared with either agent alone (36, 37). This could be approaches to modify the patient’s T cells are under achieved in a variety ways, such as by using an anti-KIR evaluation. These include engineering T cells using chi- agent (lirilumab) in combination with PD-1 or CTLA-4 meric antigen receptors (CAR) to redirect them to specific immune checkpoint inhibitors. Clinical trials are under tumor-antigen targets before reinfusion. T-cell receptor way evaluating such combinations (Table 2; ref. 12). (TCR) gene therapy is another strategy in development; the objective is to induce immune reactivity against Other immunotherapy combination partners tumors by introducing genes encoding a tumor-reactive Cytokine therapy. Cytokines have the capacity to stim- TCR into patients’ T cells, improving immune reactivity. ulate an immune response, although arguably less specif- Combining these types of approaches with other immu- ically compared with other immunotherapeutic approaches notherapies may further improve clinical efficacy. Trials (3). IL21 has a role in NK and T-cell activation, and systemic of ACT, CARs, and TCR gene therapy in combination with administration of a recombinant IL21 (rIL21) has demon- immune checkpoint inhibitors or other approaches are strated antitumor activity in tumors, including metastatic ongoing or under consideration. melanoma (38). On the basis of preclinical studies in Therapeutic vaccines. Although the various mechanisms mouse tumor models which showed enhanced antitumor of action of therapeutic vaccines are beyond the scope of this activity when rIL21 was combined with either anti–CTLA-4 review (46, 47), most vaccines are designed to (i) present or anti–PD-1 agents (39), phase I dose-escalation studies tumor antigens to the immune system and (ii) provide are evaluating these combinations in patients with immune modulation. Because of their differing mechan- advanced or metastatic melanoma (ipilimumab; ref. 40) isms of action, vaccines and other immunotherapies are or solid tumors (nivolumab; ref. 41). potential combination partners. Clinical data have shown Other cytokines are under evaluation as monotherapy for promising results with some combinations, for example, cancer therapy, but a phase II trial is ongoing with IL7, gp100 peptide vaccine and IL2 in melanoma, and ipilimu- which has a wide range of biologic activities, including a mab combined with granulocyte macrophage-colony stim- role in T-cell development, after standard therapy with ulating factor cell-based vaccine in pancreatic cancer (48, sipuleucel-T for patients with asymptomatic or minimally 49). However, no survival advantage was seen in patients symptomatic metastatic CRPC (12). with melanoma treated with gp100 plus ipilimumab versus IDO inhibition. IDO is an immunosuppressive enzyme those given ipilimumab alone in a phase III trial (13, 17). that is involved in maintaining peripheral immune toler- Various phase I and II clinical trials combining a vaccine ance by suppressing the function of both innate and adap- with a checkpoint inhibitor are ongoing in patients with

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melanoma or prostate cancer (Table 2; ref. 12). However, a nation partner and approach to treatment. Preliminary data clear demonstration of the vaccine’s ability to induce clin- for ipilimumab in combination with chemotherapy, radio- ically relevant antitumor responses in patients is required, therapy, and targeted therapy with BRAF inhibitors, are as historically, the clinical translation of cancer vaccines discussed below, alongside data with other immuno- into efficacious therapies has been challenging (with the therapies. Table 2 provides a summary of ongoing clinical exception of sipuleucel-T, the only approved therapeutic trials with immunotherapies (excluding ipilimumab) in cancer vaccine; ref. 47). Data suggest that T cells activated at combination studies with chemotherapy, radiotherapy, the vaccine site are "shut down" when they enter the tumor and targeted therapies (12). microenvironment, most likely due to tumor-mediated T- Chemotherapy combinations. Ipilimumab has shown cell–suppressive mechanisms (50, 51). With tools such as promising results when combined with chemotherapy in PD-1 immune checkpoint inhibitors that are designed to patients with melanoma and lung cancer; however, data block tumor-mediated T-cell suppression in the tumor indicate that careful consideration of the combination microenvironment, it is worth evaluating whether vaccines approach is going to be important in regard to tolerability may improve clinical efficacy when combined with a check- and optimizing patient outcomes. point inhibitor. However, data from the only published Patients with previously untreated melanoma who vaccine/PD-1 checkpoint inhibitor study showed the addi- received ipilimumab (10 mg/kg) plus chemotherapy (dacar- tion of a vaccine did not improve the efficacy of PD-1 bazine) had significantly improved OS compared with those inhibition (52). who received chemotherapy alone (11.2 months vs. 9.1 months; ref. 15). However, the benefit of the combination Integrating immunotherapies with existing relative to ipilimumab alone remains unclear, as there was therapeutic modalities not an ipilimumab-alone arm in the trial. The combination Existing treatment modalities, (e.g., chemotherapy, radio- was also less well tolerated compared with dacarbazine therapy, and molecularly targeted therapies) cause tumor alone. Grade 3 or 4 adverse events occurred in 56.3% of reduction, not only through cytotoxic/cytostatic effects, but patients treated with ipilimumab/dacarbazine compared also through mechanisms that may potentiate immune with 27.5% treated with dacarbazine/placebo (P < 0.001; activity, including modification of the tumor microenviron- ref. 15). Similarly, data from a three-arm, phase I study ment and release of tumor antigens. This activity may be showed that ipilimumab could be safety combined with complementary, even synergistic, to the immunotherapies either dacarbazine or carboplatin/paclitaxel in patients with designed to support an antitumor immune response. melanoma (68). The immune effects of chemotherapy and radiotherapy Combining ipilimumab with paclitaxel and carbopla- are widely recognized and reviewed elsewhere (53–62). tin significantly improved immune-related progression- Immune potentiating mechanisms include release of tumor free survival (irPFS) compared with chemotherapy alone antigens for immune presentation, depletion of immuno- in a phase II study in patients with NSCLC and extensive- suppressive cells (e.g., MDSCs, Tregs), activation of disease SCLC (69, 70). However, the improvement in immune effectors (NK cells, DCs, B cells, conventional irPFS was only evident when the drugs were given on a effector T cells), and sensitization of tumor cells to lysis. phased schedule (e.g., two doses of placebo plus pacli- Targeted therapies may also sensitize tumor cells to taxel/carboplatin followed by four doses of ipilimumab immune-mediated killing by a variety of mechanisms. plus paclitaxel/carboplatin), not when they were given These have been reviewed by Vanneman and colleagues concurrently. Phased ipilimumab, concurrent ipilimu- (58), and include promoting effective DC maturation, T-cell mab, and control, respectively, were associated with priming, activation, and differentiation into long-lived median irPFS of 5.7, 5.5, and 4.6 months in patients memory T cells, increasing expression of death receptors with NSCLC, and 6.4, 5.7, and 5.3 months in patients or "distress" ligands, reducing expression of prosurvival with SCLC. The overall incidence of treatment-related signals, abrogating the production of tumorigenic inflam- grade 3/4 adverse events was similar across the arms, and mation, and inhibiting immunosuppressive cell types (63). ipilimumab did not appear to exacerbate the adverse BRAF inhibitors may also increase TILs and enhance antigen events associated with chemotherapy (69, 70). Ongoing presentation (64, 65). Interestingly, while the BRAF inhi- trials are further evaluating ipilimumab/chemotherapy bitors have a potentiating effect on the immune system, combinations in melanoma and lung cancer, as well as MEK inhibitors have a possible reverse effect, reducing the in various other solid tumors, and will hopefully provide secretion of cytokines (66) and reducing the activity of information about how best to combine these treatment T lymphocytes (65) and DCs (67). modalities. Nivolumab is being investigated in combination with a Clinical experience and considerations in combining variety of agents in a large phase I trial (CheckMate 012, novel immunotherapies with existing treatment NCT01454102) in chemotherapy-na€ve patients with modalities NSCLC. Treatment arms include nivolumab monotherapy Ipilimumab is the most widely studied combination and nivolumab in combination with three, platinum-based partner for existing treatment modalities, and data highlight doublet chemotherapy regimens, bevacizumab given after the need for careful consideration in the choice of combi- at least four cycles of platinum doublet chemotherapy, and

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erlotinib (EGFR-mutation positive nonsquamous NSCLC adverse effect on response to a BRAF inhibitor. However, patients). Preliminary data indicate that nivolumab plus outcomes were poor when ipilimumab was given after platinum-based chemotherapy has a manageable safety BRAF inhibitor discontinuation (81). More data are needed, profile with no drug-related deaths reported so far. Objec- but there is some rationale to use either agent first in a tive responses have been observed in each arm, and 1-year sequencing approach, depending on the disease kinetics. In OS rates ranged from 50% to 87% (71). more rapid progressors, a BRAF inhibitor may be used first Radiotherapy combinations. Ipilimumab has been eval- to reduce tumor load followed by ipilimumab to maintain a uated in combination with radiotherapy in patients with response; in patients with more indolent disease, ipilimu- metastatic CRPC and melanoma. Promising activity with mab may be given first followed by vemurafenib to reduce manageable tolerability was observed in a phase I/II trial tumor burden (78). in patients with CRPC who had progressed after antian- In a phase I trial, concurrent administration of vemur- drogen therapy (72); however, results from a phase III afenib and ipilimumab at the approved monotherapy trial showed no significant improvement in OS with the doses or with a lower dose of vemurafenib resulted in addition of ipilimumab to radiotherapy in post-docetaxel hepatotoxicity that was greater than expected for either CRPC. A subgroup analysis did suggest benefit for agent alone (80). These safety analyses demonstrate the patients with less advanced disease (73). An analysis of risk of using vemurafenib and ipilimumab concurrently, clinical data from 21 patients with advanced melanoma and these drugs should not be used in combination who had received radiotherapy after ipilimumab progres- outside of a clinical trial. Ongoing studies are evaluating sion on the Italian Expanded Access Program indicated the optimal sequence of these agents in patients with that radiotherapy after ipilimumab treatment may further BRAF-mutant metastatic melanoma. Severe cutaneous potentiate its effect (74). A local response to radiotherapy and neurologic toxicity has also been reported in two wasdetectedin13patients(62%), while 8 patients (38%) patients with melanoma during therapy with vemurafe- did not show any local regression. The median OS for all nib after receiving treatment with a PD-1 immune check- 21 patients was 13 months (range 6–26). Eleven (85%) of point inhibitor (nivolumab or pembrolizumab; ref. 82). 13 patients with local response showed an abscopal It is also noteworthy that dose-limiting toxicities have effect, suggesting that local response to radiotherapy may been observed in patients with RCC treated with the be predictive for the abscopal response and outcome. The targeted agent sunitinib and either rhIL21 (hematologic median OS for patients with and without abscopal toxicity) or the anti–CTLA-4 agent tremelimumab (renal responses was, respectively, of 22.4 months (range 2.5– failure), further emphasizing the need for caution when 50.3) and 8.3 months (range 7.6–9.0). There are now evaluating combinations (83, 84). over 15 clinical trials alone in progress to evaluate ipili- RCC is a tumor for which combining immunotherapy mumab plus radiotherapy. and targeted therapy is of substantial interest. Preliminary Initial data from a phase I trial of MPDL3280A, an anti– data from a phase II trial of nivolumab in combination with PD-L1 monoclonal antibody, in combination with local pazopanib or sunitinib in patients with metastatic RCC radiotherapy showed evidence of activity in the five patients showed evidence of activity with ORRs of 45% and 52%, treated (75). Overall, case reports and data from several respectively, and a manageable safety profile (85). This trial small clinical studies showing successful, sometimes dra- and others evaluating various combinations in RCC matic, outcomes with radiotherapy/immunotherapy com- continue. binations in patients with melanoma provide additional The anti-CD137 agents urelumab and PF-05082566 are support for further evaluation; these are comprehensively both in phase I trials in combination with rituximab in discussed by Barker and Postow (76). patients with non-Hodgkin lymphoma (Table 2). Clinical Targeted therapy combinations. Clinical data are lim- study of these agents with rituximab is based on preclin- ited on the efficacy of combining ipilimumab with tar- ical data that have shown enhanced tumor regression geted agents, although numerous trials are ongoing, when an anti-CD137 agent was used after a therapeutic particularly in melanoma, where three targeted therapies monoclonal antibody (86, 87). The anti-CD137 antibody are now approved in the United States for patients with is proposed to enhance rituximab-dependent cytotoxicity melanoma and mutated BRAF (dabrafenib, vemurafenib, through antigen-dependent cell-mediated cytotoxicity and trametinib). (86). Recent preclinical data showing enhanced antilym- Immunotherapy and BRAF inhibitor combinations are phoma activity with rituximab combined with KIR block- extensively reviewed by Hu-Lieskovan and colleagues (77). ade (lirilumab) also support clinical investigation of this Some data indicate that the sequencing of BRAF inhibitors combination (34). and ipilimumab has a marked effect on the efficacy and tolerability of the combination in patients with BRAF- Conclusions mutant melanoma, and indicate that the drugs should be Immuno-oncology is an evolving treatment modality, sequenced (78–80). Data from a recent retrospective anal- with agents being studied for their potential to provide ysis of a cohort of patients treated with immunotherapy and long-term survival across a broad range of tumor types, then a BRAF inhibitor (with or without a MEK inhibitor) and for their synergistic activity when combined with other showed prior immunotherapy did not appear to have an treatment modalities. It is important now to determine how

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to advance this field and how to use these new immu- Disclosure of Potential Conflicts of Interest notherapies most effectively to achieve the best patient S.J. Antonia is a consultant/advisory board member for Bristol-Myers Squibb and MedImmune/AstraZeneca. J. Larkin reports receiving com- outcomes. Areas of investigation are broad, and include mercial research grants from Bristol-Myers Squibb, Novartis, and Pfizer, combining or sequencing immunotherapies that target and is a consultant/advisory board member for Bristol-Myers Squibb, distinct immune pathways, combining or sequencing an GlaxoSmithKline, Merck, Novartis, Pfizer, and Roche/Genentech. (Note: all consultancy/advisory board membership was uncompensated after immunotherapeutic agent with existing treatment modal- 2012.) P.A. Ascierto reports receiving commercial research grants from ities, and determining the optimal schedule of therapies in Bristol-Myers Squibb, Merck, Roche/Genentech, and Ventana; speakers bureau honoraria from Bristol-Myers Squibb, GlaxoSmithKline, and combination regimens. At present, it is difficult to identify Roche/Genentech; and is a consultant/advisory board member for Bris- the best combination approaches to pursue given the lim- tol-Myers Squibb, GlaxoSmithKline, Merck, Novartis, Roche/Genentech, ited data and the somewhat unexpected occurrence of and Ventana. No other potential conflicts of interest were disclosed. toxicity with some combinations (e.g., ipilimumab and Authors' Contributions vemurafenib). Future data from preliminary clinical studies Conception and design: J. Larkin, P.A. Ascierto will help to direct research. Development of methodology: J. Larkin, P.A. Ascierto Combining immunotherapies has the potential to over- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P.A. Ascierto come more than one of the barriers that tumor cells develop Analysis and interpretation of data (e.g., statistical analysis, biosta- to evade the immune system, and may provide an OS tistics, computational analysis): J. Larkin, P.A. Ascierto benefit in a greater portion of patients compared with either Writing, review, and/or revision of the manuscript: S.J. Antonia, J. Larkin, P.A. Ascierto agent alone (Fig. 1). However, the ideal sequence, schedule, Administrative, technical, or material support (i.e., reporting or orga- and combination of immunotherapies need to be deter- nizing data, constructing databases): P.A. Ascierto mined. Likewise, it is important to determine optimal dose, Study supervision: P.A. Ascierto schedule, and sequence when combining an immunother- Acknowledgments apy with radiotherapy, chemotherapy, or targeted agents, as The authors thank Rebecca Turner of StemScientific, a health care com- these therapies all have different mechanisms of action. A munications firm funded by Bristol–Myers Squibb, for providing writing and editorial support. final consideration for combining immunotherapies will be to identify the regimens with the best risk–benefit profile. Grant Support We can expect improvements in overall clinical efficacy as J. Larkin is supported by the NIH Research Royal Marsden Hospital/ new agents targeting alternative or overlapping tumor-asso- Institute of Cancer Research Biomedical Research Centre for Cancer. ciated immunosuppressive mechanisms are developed and Received July 2, 2014; revised September 17, 2014; accepted October 4, used in combination or sequentially. 2014; published OnlineFirst October 23, 2014.

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Immuno-oncology Combinations: A Review of Clinical Experience and Future Prospects

Scott J. Antonia, James Larkin and Paolo A. Ascierto

Clin Cancer Res Published OnlineFirst October 23, 2014.

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