Propelling Immunotherapy Combinations Into the Clinic

Immune checkpoint inhibitors produce durable long-term survival in some patients with advanced melanoma and lung cancer. Better immune targets and combination strategies can harness the immune system by supporting the three elements of a successful T-cell antitumor response: (A) generation of sufficient numbers of antitumor T cells within the lymphoid compartment; (B) effective T-cell trafficking and extravasation out of the lymphoid compartment, through the bloodstream, and into the tumor microenvironment; and (C) T-cell effector function within the tumor microenvironment that is characterized by the ability to bypass immune checkpoints, soluble and metabolic inhibitory factors, and inhibitory cells. Strategies that hold promise include dual immune checkpoint blockade, as well as the combination of immune checkpoint blockade with costimulatory receptor agonists, enhancers of innate immunity, inhibition of indoleamine 2,3-dioxygenase, adoptive T-cell transfer/T-cell engineering, therapeutic vaccines, small-molecule inhibitors, and radiation therapy. Novel, rational clinical trial designs seek to combine targeted agents and one or more immune checkpoint inhibitors, with the goal of producing deep and durable antitumor responses, which thus far have been observed in only a minority of patients.

Introduction

Novel immunotherapy combinations raise the prospect of improving both overall survival (OS) and quality-of-life outcomes for cancer patients. Immune checkpoint blockade targeting programmed death ligand 1 (PD-L1); its receptor, programmed death 1 (PD-1); and/or cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) causes durable tumor regression, most notably in melanoma and lung cancers, and has demonstrated favorable activity in bladder cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, and hematologic malignancies.[1-8] This review summarizes promising new targets and immunotherapy combination strategies currently under clinical development.

A Historical Perspective

A century ago, in the first attempts at immunotherapy, Dr. William Coley inoculated cancer patients with dead bacteria and observed several spontaneous remissions.[9] For decades, cytokines such as interleukin (IL)-2 (aldesleukin) and interferon alfa have benefited small subsets of otherwise healthy patients with RCC and melanoma.[10-13] Sipuleucel-T, an autologous active therapeutic vaccine that induces T-cell immune responses against prostatic acid phosphate antigen, improved survival and was approved in 2010 by the US Food and Drug Administration (FDA) for patients with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer.[14] Ipilimumab, a monoclonal antibody targeting the CTLA-4 immune checkpoint, improved survival and was approved by the FDA in 2011 for treatment of melanoma.[6,15,16] In 2014, pembrolizumab and nivolumab became the first PD-1 checkpoint inhibitors to be approved for use in melanoma among patients with ipilimumab and BRAF inhibitor–refractory disease.[17,18] Nivolumab was approved in March 2015 for platinum-refractory, metastatic, squamous-cell non–small-cell lung cancer (NSCLC), based upon improved survival when compared with conventional chemotherapy with docetaxel; it was approved for nonsquamous NSCLC in October.[19,20] On September 30, 2015, the FDA granted accelerated approval of nivolumab combined with ipilimumab for BRAF V600 wild-type unresectable or metastatic melanoma, based on randomized controlled data showing improved response rate and progression-free survival (PFS) as well as prolonged duration of response with the combination vs ipilimumab alone (as described in the nivolumab package insert). Nivolumab plus ipilimumab is the first checkpoint inhibitor combination to be approved by the FDA. Based on a favorable response rate, pembrolizumab was approved in October 2015 for platinum-refractory and, if applicable, tyrosine kinase inhibitor (TKI)-refractory, metastatic NSCLC that is PD-L1–positive based on an FDA-approved test (as described in the pembrolizumab package insert).[1] Figure 1 outlines cancer immunotherapy milestones of the last two decades.

The Cancer Immunity Cycle Offers Therapeutic Targets

An emerging hallmark of cancer is immunoevasion-the cancer cell’s ability to avoid destruction by the immune system.[21] The three general categories of immunoevasive mechanisms include: (A) an insufficient number of T cells generated within the lymphoid compartment; (B) an insufficient number of T cells extravasating into the tumor; and (C) inhibition of T cells in the tumor microenvironment (Figure 2). The tumor microenvironment, in turn, offers three main immunoevasive tools: (1) surface membrane proteins that function as immune checkpoints, including PD-1, CTLA-4, lymphocyte-activation gene 3 (LAG-3) protein, T-cell immunoglobulin and mucin domain–containing protein 3 (TIM-3), B- and T-lymphocyte attenuator (BTLA), and the adenosine A2a receptor (A2aR); (2) the relationship between selected soluble factors and metabolic alterations, such as IL-10, transforming growth factor beta, adenosine, indoleamine 2,3-dioxygenase (IDO), and arginase; and (3) inhibitory cells, including cancer-associated fibroblasts (CAFs), regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages.

Immunotherapeutic strategies are under development to disrupt steps in the above-described immunoinvasive mechanisms A to C and generate a tumor-specific immune response (Table 1 and Figure 2). For example, CTLA-4 blockade (immune checkpoint inhibition), agonism of T-cell costimulatory receptors, adoptive T-cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs), and T-cell engineering with chimeric antigen receptor (CAR) T-cell therapy or T-cell receptor gene therapy all increase the antitumor T-cell number in the lymphoid compartment.[22] Radiation, chemotherapy, cytokines, and certain molecularly targeted therapies all increase the influx of T cells into tumors.[22] Additional approaches to boost tumor-targeted immune responses include immune checkpoint inhibition (anti–PD-1, anti–PD-L1, anti–LAG-3, anti-A2aR); suppression of T regulatory cells (Tregs) and MDSCs; and stimulation of innate immune cells, such as natural killer (NK) cells, macrophages, and dendritic cells.

Combining immune checkpoint inhibitors with other immunotherapeutics

Either combining or sequencing immunotherapies with distinct targets is a rational approach for improving efficacy. Combination approaches currently under clinical development include dual checkpoint inhibition, checkpoint inhibition plus agonism of T-cell costimulatory receptors, and checkpoint inhibition plus TIL ACT-strategies that may simultaneously exploit expansion of T cells in the lymphoid compartment (anti–CTLA-4) and antitumor T-cell function at the tumor site (see Figure 2, parts A and C). Immune checkpoint blockade is also being combined with radiation, chemotherapy, and small-molecule inhibitors, acting at the level of trafficking and effector function (see Figure 2, parts B and C). Other combinations focus entirely on the tumor microenvironment. Examples include use of checkpoint blockade plus suppressors of Tregs and MDSCs; and checkpoint blockade plus stimulation of the innate immune cells, such as NK cells, macrophages, and dendritic cells (see Figure 2, part C).

Dual T-cell checkpoint blockade

CTLA-4 (also known as CD152) is expressed on the surface of cytotoxic T cells and competes with the costimulatory receptor CD28 to bind to B7-1 (CD80) or B7-2 (CD86) on the antigen-presenting cell. Signaling through CTLA-4 produces an inhibitory signal in T cells. Blocking CTLA-4 with an antibody prevents this negative signaling in tumor cell–specific T cells within lymph nodes, expanding the number of antitumor T cells as well as the breadth of their repertoire.[23-25] If the T cell is primed and migrates to the site of effector function, then it must still go on to evade the tumor’s immune checkpoint ligands.[26] One such checkpoint ligand is PD-L1, which binds to the T-cell surface checkpoint protein PD-1. When PD-1 on the effector T cell interacts with PD-L1 on either the tumor cell or the myeloid cells, immune tolerance develops.[27] When this interaction is blocked, the T cell is freed to perform its function.[26,28,29] Dual blockade with both CTLA-4 inhibition and PD-1/PD-L1 inhibition targets immune evasion at the site of both priming/activation (lymph nodes) and effector function (tumor parenchyma).

Single-agent blockades of CTLA-4, PD-1, and PD-L1 have been active across several tumor types.[7,8,16,18] In melanoma, the antitumoral activity of checkpoint inhibition has translated into improved long-term survival. A phase III study of ipilimumab in patients with metastatic melanoma showed an OS rate of 25% at 2 and 3 years.[30] A pooled analysis of 12 ipilimumab trials showed that the OS plateau of 22% began at 3 years and extended up to 10 years in some patients.[31] Immunotherapy combinations offer the prospect of long-term survival for increased numbers of patients with melanoma and other cancers.

The first clinical data to support dual checkpoint blockade came from a phase I study of ipilimumab (1 mg/kg or 3 mg/kg) and nivolumab (0.3 mg/kg, 1 mg/kg, or 3 mg/kg) either concurrently or sequentially in patients with stage III or IV melanoma.[6] In the combination group, the objective response rate (ORR) was 40%, with grade 3/4 treatment-related adverse events occurring in 53% of patients. At the maximum tolerated dose (MTD) (nivolumab at 1 mg/kg and ipilimumab at 3 mg/kg), the ORR was 53% and tumor volume reductions of 80% or more were observed among the responders. The responses appeared to be more rapid and deeper than those reported previously. In the sequential therapy group, the ORR was 20%, and 18% of patients had grade 3/4 adverse events related to therapy.

A phase III trial (CheckMate 067) is evaluating concurrent ipilimumab and nivolumab vs monotherapy with either drug in patients with untreated, unresectable stage III or IV melanoma. The median PFS was superior in both the combination arm (11.5 months [95% confidence interval (CI), 8.9–16.7]; hazard ratio [HR] for disease progression or death, 0.42 [99.5% CI, 0.31–0.57; P < .001]) and the nivolumab arm (6.9 months [95% CI, 4.3–9.5]; HR, 0.57 [99.5% CI, 0.43–0.76; P < .001]), when compared with the ipilimumab arm (2.9 months [95% CI, 2.8–3.4]). The study was not designed to detect statistical differences between the nivolumab-containing arms. In patients with PD-L1–positive tumors, the PFS was 14 months in both the combination and nivolumab groups. However, in patients whose tumors did not express PD-L1, the combination group had an improved PFS compared with patients treated with nivolumab alone: 11.2 months (95% CI, 8.0 to not reached) vs 5.3 months (95% CI, 2.8–7.1). Grade 3/4 adverse effects related to therapy occurred in 16.3% of patients in the nivolumab group, 27.3% of patients in the ipilimumab group, and 55.0% of patients in the combination group. Longer follow-up is needed to determine the impact on OS.

A phase I, open-label, dose-escalation and expansion study evaluating durvalumab and tremelimumab in advanced solid tumors showed a 27% response rate (95% CI, 13–46) in PD-L1–negative patients, with a disease control rate of 48% (95% CI, 31–66) at ≥ 16 weeks after therapy.[32] Forty percent of patients experienced grade 3/4 toxicities, most frequently colitis, and 20% of patients had to discontinue treatment due to drug-related adverse events. Increasing doses of tremelimumab were associated with higher rates of serious adverse events. Durvalumab at 20 mg/kg every 4 weeks plus tremelimumab at 1 mg/kg every 4 weeks was the dose level chosen for phase III development. The phase I study showed that at this dose level, toxicity leading to discontinuation was < 10%, but lower tremelimumab dosing did not erode clinical efficacy. Notably, anti–PD-1/PD-L1 monotherapy produces an approximately 5% to 10% response rate in PD-L1–negative patients; thus, the addition of low-dose anti–CTLA-4 therapy may benefit these patients.[1]

Although small-cell lung cancer (SCLC) is chemosensitive, responses to both first- and second-line chemotherapy are short-lived and outcomes remain poor.[33,34] CheckMate 032 (ClinicalTrials.gov identifier: NCT01928394) is an ongoing, open-label, phase I/II study of nivolumab with or without concurrent ipilimumab in patients with recurrent SCLC. Patients were randomized to two arms: nivolumab at 3 mg/kg every 2 weeks or nivolumab plus ipilimumab in three dose cohorts (1 mg/kg + 1 mg/kg, 1 mg/kg + 3 mg/kg, or 3 mg/kg + 1 mg/kg) every 3 weeks for 4 cycles, followed by maintenance nivolumab (3 mg/kg every 2 weeks). The primary endpoint was ORR by Response Evaluation Criteria In Solid Tumors (RECIST) v1.1. Grade 3/4 drug-related adverse events occurred in at least 5% of patients and included diarrhea and rash (6% each in the combination arm). Pneumonitis occurred in one patient per arm, and one patient died as a result of drug-related myasthenia gravis.

Updated interim data for 40 patients in the nivolumab arm and 46 patients in the combination arm were presented in June 2015, and showed an ORR of 32.6% in the nivolumab-plus-ipilimumab arm vs 18% in the nivolumab arm. One patient in the nivolumab plus ipilimumab arm had a complete response. Responses occurred in patients regardless of PD-L1 expression, and there was a trend towards increased OS in the nivolumab-plus-ipilimumab arm (8.2 months [95% CI, 3.7 to not reached]) vs the nivolumab-alone arm (4.4 months [95% CI, 2.9–9.4]).[35] The combination of nivolumab and ipilimumab is also being studied in other cancers (Table 2), including a phase I study in metastatic RCC (ClinicalTrials.gov identifier: NCT01472081), with interim results showing an ORR of 29% (6 of 21 patients) in the group receiving nivolumab at 3 mg/kg plus ipilimumab at 1 mg/kg, and an an ORR of 39% (9 of 23 patients) in the group treated with nivolumab at 1 mg/kg plus ipilimumab at 3 mg/kg.[36]

In September 2015, updated results from CheckMate 012 evaluating the safety and efficacy of first-line nivolumab monotherapy vs nivolumab-based combinations in advanced NSCLC showed that nivolumab at 3 mg/kg every 2 weeks plus ipilimumab at 1 mg/kg every 12 weeks resulted in a response rate of 39% (n = 38) and a disease control rate of 74% (95% CI, 57–87).[37] There was a very low frequency of treatment-related grade 3/4 adverse events leading to discontinuation in this dose cohort, again indicating that low-dose, less frequent anti–CTLA-4 therapy combined with anti–PD-1 therapy may produce optimal clinical results, even in the first-line setting.

Another T-cell surface checkpoint protein is the A2aR. The Warburg effect, a metabolic abnormality in which cancer cells exhibit increased rates of glycolysis and lactic acid fermentation; hypoxia in the tumor microenvironment; and CD73 on both tumor cells and CAFs all lead to elevated adenosine levels in the tumor milieu.[38] Adenosine signaling via the A2aR directly inhibits T cells in addition to supporting the growth of CAFs, which directly inhibit killing of tumor cells by CD8+ T cells (cytotoxic T cells). PBF-509, an A2aR antagonist entering phase I testing in NSCLC (ClinicalTrials.gov identifier: NCT02403193), will be studied in combination with other checkpoint inhibitors in the near future.

Phase I studies are currently evaluating a host of dual checkpoint blockade combinations, including ipilimumab plus nivolumab, ipilimumab plus pembrolizumab (anti–PD-1), tremelimumab (anti–CTLA-4) plus durvalumab (anti–PD-L1), and the anti–LAG-3 monoclonal antibody BMS-986016 plus nivolumab (see Table 2).[39,40]

T-cell checkpoint blockade plus costimulatory receptor agonists

Checkpoint inhibition combined with costimulatory receptor agonists may be an opportunity for synergy, but to date this remains theoretical. Selection of costimulatory agonists must be undertaken with cautious optimism, as evidenced by the excessive toxic effects of the CD28 agonist TGN1412 in a first-in-human phase I trial.[41] Six healthy volunteers, 18 to 30 years old, were treated with 0.1 mg/kg of TGN1412, and within 90 minutes each experienced a cytokine storm leading to multi-organ failure and prolonged intensive care. Given that CD28 is expressed on all mature T cells, this antibody agonist was thought to have a “super-agonist” effect.[42] Other costimulatory molecules are expressed on subgroups of T cells and have shown less propensity to cause such dramatic adverse events.

Clinical studies targeting costimulatory receptors with agonistic antibodies are currently in development. CD137 (also called 4-1BB and TNFRSF9 [tumor necrosis factor receptor superfamily, member 9]), glucocorticoid-induced TNFR family-related protein (GITR, also known as TNFRSF18 [TNFR superfamily, member 18]), and CD134 (also called OX40 and TNFRSF4 [TNFR superfamily, member 4]) are costimulatory receptors that promote T-cell proliferation and survival. CD40 (also known as TNFRSF5 [TNFR superfamily, member 5]) stimulates the activation of antigen-presenting cells. CD27 is a costimulatory receptor implicated in multiple functions of T cells, NK cells, and B cells, including long-term memory.[43]

A single-arm, open-label, phase I trial evaluated combined treatment with the CD40 agonist antibody CP-870,893 plus tremelimumab in 24 patients with metastatic melanoma.[44] The ORR was 27.3% and median OS was 26.1 months. The MTD level was CP-870,893 at 0.2 mg/kg every 3 weeks plus tremelimumab at 10 mg/kg every 12 weeks. Two patients (9.1%) had complete responses. Grades 1 and 2 cytokine release syndrome occurred in 19 patients (79.2%) immediately after the administration of CP-870,893, but symptoms resolved within 24 hours with standard supportive care. A host of early-phase checkpoint blockade plus costimulatory receptor agonist clinical trials are underway (see Table 2).

T-cell checkpoint blockade to improve innate immune cell function

Killer cell immunoglobulin-like receptors (KIRs) are cell surface proteins found on NK cells that function to downregulate NK cell activity. In immune physiology, KIRs bind to major histocompatibility complex (MHC) class I molecules expressed on all normal cells to maintain self-tolerance. However, tumor cells also express MHC class I antigens and thereby evade immune surveillance by NK cells. Anti-KIR antibodies can block this immunoevasion, thereby potentiating NK cell effector function and thus innate immunity.[45-48] Lirilumab is an inhibitory anti-KIR antibody that is being studied in combination with ipilimumab in solid tumors and with nivolumab in hematologic malignancies.[49] Given the close interplay between the adaptive and innate immune systems, strategies that enhance cellular components of both response systems may prove efficacious.

Checkpoint blockade plus IDO inhibition

IDO is a heme-containing redox enzyme that suppresses the priming and activation of the adaptive immune response through catabolism of tryptophan in the tumor-draining lymph nodes and tumor microenvironment. Depletion of tryptophan leads to impaired activation of helper and cytotoxic T cells, allowing for control of autoimmunity and maintenance of placental pregnancy.[50-52] Preclinical data have shown that this immunosuppressive mechanism can be co-opted by tumors.[53-55] These data have led to the development of multiple IDO pathway inhibitors, including indoximod, epacadostat (INCB24360), and, most recently, GDC-0919. Indoximod differs from the other two compounds in that it does not efficiently directly inhibit IDO, but rather acts downstream of the enzyme to reduce its effects in the tumor microenvironment.[56] Melanoma mouse models have shown that IDO represses the antitumor effect of anti–CTLA-4 therapy. Blockade of IDO and CTLA-4 reversed this effect and increased antitumor activity.[57,58] Preliminary data from a phase I/II study of this combination have been promising, and blockade of IDO and PD-1 has shown significant clinical activity.[59] Other combination therapies include IDO inhibitors administered along with therapeutic cancer vaccines, chemotherapy, and immune checkpoint–blocking drugs (ClinicalTrials.gov identifiers: NCT02327078, NCT02178722, NCT02318277, NCT02298153, NCT01604889, NCT02073123, and NCT02471846) in phase I and II clinical trials (see Table 2). There is work underway to target a related enzyme, tryptophan 2,3-dioxygenase, which is also upregulated in tumors and may act in a similar fashion to IDO.[60]

Checkpoint blockade plus adoptive T-cell transfer/T-cell engineering

ACT is the process by which TILs are grown out of tumors harvested from patients, expanded in vitro, and reintroduced into the patient.[61] Prior to reinfusion into the patient, CAR T-cell therapy can be used to direct the T cell to specific tumor antigens. A third approach is T-cell receptor gene therapy, in which genes encoding tumor-reactive T-cell receptors are introduced into the patient’s T cells.

In a phase II study, ACT of melanoma TILs plus IL-2 led to complete response in 20 of 93 patients (22%) with metastatic, largely pretreated melanoma; 19 patients had durable complete responses lasting at least 3 years after treatment.[62] However, the lymphodepleting preparative regimen (chemotherapy with or without total body irradiation) caused toxicity, including a treatment-related death in a patient who developed sepsis due to an undetected diverticular abscess. A variety of cancer/testis antigens, such as New York–esophageal cancer–1 (NY-ESO-1) protein and melanoma antigen gene family (MAGE)-A10, have been used to generate tumor-specific T-cell receptors and have shown promising results in melanoma and synovial cell sarcoma.[63] CAR T-cell therapy directed at CD19 and mesothelin has shown positive results in multiple myeloma and solid malignancies, respectively.[64,65] Despite promising early data, toxicity reports included a patient with metastatic colorectal cancer who developed respiratory distress within 15 minutes of treatment with CAR T cells targeting epidermal growth factor receptor 2 (EGFR2; also known as human epidermal growth factor receptor 2 [HER2] and erb-b2 receptor tyrosine kinase 2 [ERBB2]).[66] The patient had multi-organ failure and multiple cardiac arrests in the context of a cytokine release storm, which led to death. One patient treated intermittently with CAR T-meso cells, which express a murine antibody to human mesothelin, developed fatal anaphylaxis and cardiac arrest after the third infusion.[67] CARs are being developed with different antibody fragments and more conservative dose-escalation methods to mitigate toxicities.[68] Unexpected toxicity was also encountered after two patients treated with autologous anti–MAGE-A3 T-cell receptor–engineered T cells developed comas and then died of progressive necrotizing leukoencephalopathy due to previously unrecognized expression of MAGE-A3 in the brain.[69] Early-phase studies utilizing ACT combined with immune checkpoint inhibition are underway, and special care must be taken to attenuate the potential toxicities of these combinations (see Table 2; ClinicalTrials.gov identifier: NCT02210104).

Checkpoint Blockade Plus Small Molecules That Create an Immune-Active Microenvironment

Checkpoint blockade plus histone deacetylase inhibition

Increased tumor expression of T-cell chemokines, such as chemokine (C-C motif) ligand 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10), is associated with a better response to immunotherapy, and is strongly and positively associated with increased T-cell infiltration and improved patient survival.[70-72] Therefore, enhancement of expression of T-cell chemokines by use of small-molecule biologic effectors such as histone deacetylase (HDAC) inhibitors may augment response to PD-1 blockade immunotherapy. HDAC inhibition has generally led to disappointing results when used alone and in combination with chemotherapy in solid tumors. However, recent preclinical studies indicate that HDAC inhibition may contribute to an immune-active environment, making tumor cells more susceptible to anti–PD-1 therapy. Mouse tumor models indicate a synergistic interaction between anti–PD-1 and the HDAC inhibitor, which is entirely T-cell dependent; a phase I/II trial has been designed that will combine an HDAC inhibitor with immune checkpoint blockade.[73]

In a phase I/II trial of 5-azacytidine in combination with entinostat in 19 patients with advanced NSCLC, one patient had a complete response and another had a partial response. Four patients had partial responses after subsequent therapy; one of those patients was treated with anti–PD-1.[74] In a phase I study, five patients were treated with a combination of 5-azacytidine and entinostat prior to being treated with anti–PD-1 or anti–PD-L1 therapy. Three patients had partial responses and two had stable disease by RECIST.[75] Rationally designed clinical trials utilizing concurrent HDAC inhibitors and immune therapy with engrained biomarker analysis will be underway soon (see Table 2).

Checkpoint blockade plus EGFR TKIs

The relationship between EGFR-sensitizing mutations and response to checkpoint blockade is not clear. Patients with metastatic NSCLC with EGFR-sensitizing mutations treated with pembrolizumab had an ORR of 7.8% (95% CI, 2.9–16.2) vs 21.6% (95% CI, 17.8–25.6) in EGFR wild-type tumors.[76] In addition, a retrospective analysis demonstrated that 0 of 22 patients with EGFR-sensitizing mutations responded to pembrolizumab after prior TKI therapy, whereas patients who were TKI-naive had higher response rates.[77] In vitro experiments showed that PD-L1 expression decreased in TKI-responsive cell lines after treatment with a TKI. A phase I study of the combination of erlotinib and nivolumab (3 mg/kg) in chemotherapy-naive patients with advanced EGFR-mutated NSCLC showed that 3 of 20 patients (15%) with acquired erlotinib resistance had a partial response, with 9 of 20 patients having stable disease.[78] These results may have implications in future clinical trial designs that incorporate checkpoint inhibition in EGFR-mutated NSCLC; clinical studies are ongoing (see Table 2; ClinicalTrials.gov identifiers: NCT02364609 and NCT02039674). Third-generation EGFR TKIs (AZD9291 and rociletinib) have shown promise in tumors harboring the T790M gatekeeper mutation; however, the recent suspension of clinical trials combining AZD9291 and durvalumab due to cases of lung toxicity may portend difficulties combining these agents.[79-81]

Checkpoint blockade plus VEGF inhibition

Tumor cells secrete vascular endothelial growth factor (VEGF) A, which has immunosuppressive effects via support of the formation of MDSCs, decreased T-cell priming, and decreased dendritic cell costimulatory molecule expression. Inhibition of VEGF or its receptor via antibodies such as bevacizumab or small molecules like sunitinib has immunomodulatory effects that include suppression of MDSCs and Tregs and downregulation of immunosuppressive signal-transduction pathways.[43,82] A phase I study demonstrated that concurrent administration of ipilimumab and bevacizumab was safe and feasible in patients with metastatic melanoma, showing a 2-year median OS of 25.1 months.[83] Many ongoing studies are evaluating the combination of immune checkpoint inhibition and VEGF or VEGF receptor inhibition in RCC, gastric cancer, and NSCLC (ClinicalTrials.gov identifiers: NCT01472081, NCT01984242, NCT02210117, NCT02572687, and NCT02443324).

Checkpoint blockade plus BRAF V600E inhibition

Vemurafenib is a potent inhibitor of mutant BRAF; it has immunomodulatory effects, including increasing expression of MHC class I molecules and differentiation antigens of melanocytes (gp100, MART1, tyrosinase, and others), while decreasing secretion of immunosuppressive cytokines.[43,82,84] Combining immune checkpoint inhibitors with BRAF inhibitors has led to unexpected toxicity. Vemurafenib was combined with ipilimumab in a small phase I study of patients with BRAF-mutated metastatic melanoma, which was halted due to grade 3 elevation of liver enzymes in 6 of 10 patients.[85] A report of 13 patients treated sequentially with ipilimumab then vemurafenib showed that the 3 patients who received vemurafenib within 4 weeks of the last dose of ipilimumab rapidly developed a grade 3 rash, which was biopsy-proven to be a drug hypersensitivity reaction.[86] All three patients resumed treatment after the vemurafenib was suspended for up to 11 days and restarted at a lower dose. Two patients treated with vemurafenib after treatment with anti–PD-1 therapy (nivolumab and pembrolizumab) developed hypersensitivity reactions with multi-organ involvement, with one patient also developing acute inflammatory demyelinating polyneuropathy.[87] A phase I study (ClinicalTrials.gov identifier: NCT02027961) evaluating the combination of durvalumab (anti–PD-L1) with dabrafenib (a BRAF inhibitor) and/or trametinib (a MEK inhibitor) in patients with BRAF-mutated and BRAF wild-type advanced melanoma did not reach an MTD and showed clinical activity across all cohorts.[88] Three targeted therapies-vemurafenib, dabrafenib, and trametinib-are FDA-approved for patients with BRAF-mutated advanced melanoma, and studies combining these and others (such as the MEK inhibitor cobimetinib) with immune checkpoint inhibitors are ongoing (ClinicalTrials.gov identifiers: NCT02357732, NCT01656642, and NCT02130466).

Checkpoint Blockade Plus Therapeutic Cancer Vaccines

Sipuleucel-T is the solitary approved therapeutic cancer vaccine. Antigen-specific immunotherapy via cancer vaccines has generally lacked clinical efficacy.[89-96] This may be due to tumoral immunosuppressive mechanisms that inactivate T cells in the tumor microenvironment.[97,98] Immune checkpoint inhibition could theoretically allow vaccine-primed and -activated T cells to accomplish effector functions in the tumor microenvironment. One study showed no benefit of adding a multipeptide vaccine to nivolumab in refractory melanoma.[99] Based on preclinical and clinical data demonstrating possible synergy of anti–CTLA-4 administered in combination with tumor cell vaccines that produce granulocyte-macrophage colony-stimulating factor, ipilimumab plus systemic sargramostim was compared with ipilimumab alone in patients with unresectable stage III or IV melanoma.[100,101] The combination had lower toxicity and yielded better OS (17.5 months vs 12.7 months; P = .01), without any difference in PFS.[102] Several phase I and II combination studies utilizing vaccines and PD-1 inhibitors are ongoing, and the topic is reviewed in detail elsewhere.[103]

Checkpoint Blockade Plus Chemotherapy

Numerous ongoing trials are evaluating the combination of chemotherapy and checkpoint blockade in solid tumors, including melanoma, NSCLC, and SCLC. In untreated metastatic melanoma, a phase III study showed that ipilimumab (at 10 mg/kg) plus dacarbazine improved OS compared with dacarbazine alone (11.2 vs 9.1 months, respectively), but this was at the expense of higher toxicity and there was no ipilimumab-alone comparator arm.[15] A phase II study showed that phased but not simultaneous ipilimumab plus platinum doublet chemotherapy (carboplatin/paclitaxel) improved immune-related PFS in patients with stage IIIB or IV NSCLC and extensive-stage SCLC, when compared with chemotherapy alone.[104,105] The choice of chemotherapy and dosing schedule are thus critical to optimizing outcomes of checkpoint blockade and chemotherapy combinations. With this in mind, a phase I four-cohort study evaluated first-line nivolumab at 10 mg/kg (N10) vs 5 mg/kg (N5) in combination with gemcitabine/cisplatin (N10) in advanced squamous-cell NSCLC, pemetrexed/cisplatin (N10) in advanced nonsquamous NSCLC, and paclitaxel/carboplatin (N5 vs N10) in combined cohorts of squamous and nonsquamous NSCLC.[106] The toxicity profile was additive, representing effects of both nivolumab and chemotherapy. The ORR, PFS, and 1-year OS outcomes were acceptable. In particular, the 1-year OS rate was 85% for the N5 paclitaxel/carboplatin group and 87% for the N10 pemetrexed/cisplatin group, which may reflect a positive signal.

A phase Ib study enrolled untreated patients with locally advanced or metastatic NSCLC to three treatment arms of atezolizumab plus chemotherapy, including carboplatin/pemetrexed, carboplatin/paclitaxel, and carboplatin/nab-paclitaxel.[107] Atezolizumab at 15 mg/kg every 3 weeks was administered with standard chemotherapy for 4 to 6 cycles followed by atezolizumab maintenance or atezolizumab/pemetrexed maintenance in the carboplatin/pemetrexed arm. An interim analysis showed that the ORR was 67% (95% CI, 48–82) by RECIST, with the carboplatin/pemetrexed arm having the highest response rate at 75% (95% CI, 45–93). The only two complete responses occurred in the carboplatin/nab-paclitaxel arm. The toxicity profile was as expected for chemotherapy, and no pneumonitis was observed. There was one grade 5 adverse event in a patient in the carboplatin/nab-paclitaxel arm who developed candidemia after prolonged neutropenia. Overall, the combination therapy response rates exceeded the 30% traditionally expected with platinum doublet chemotherapy; more mature data are forthcoming.

Checkpoint Blockade Plus Radiation Therapy

Recent studies indicate that at least one component of radiation-induced tumor control involves activation of the adaptive immune system as a result of tumor antigen release following radiation therapy.[108-110] Combining radiation therapy with immune checkpoint blockade may be an effective approach to stimulation of the adaptive immune system, with further amplification of immune response achieved via systemic immune checkpoint blockade. Preclinical studies have shown that combining radiation therapy with immune checkpoint blockade offers an opportunity for synergistic response rates.[111-114] The abscopal effect of radiation therapy is a poorly understood phenomenon that refers to a systemic antitumor response incited by localized radiation. Case reports and series have described the abscopal effect in patients with advanced melanoma treated with ipilimumab and radiation therapy.[115,116] A recent phase I clinical study combining ablative radiation therapy with ipilimumab in patients with metastatic melanoma showed excellent local control at the site of radiation and an 18% partial response rate at sites outside the radiation treatment field.[114] Multiple ongoing and upcoming phase I/II clinical studies that include patients with lung cancer, melanoma, and other solid tumors aim to evaluate whether the combination of radiation therapy with immune checkpoint blockade will be an effective approach for improving response rates in the metastatic setting (ClinicalTrials.gov identifiers: NCT02221739, NCT02239900, NCT02463994, NCT02383212, and NCT02444741) and improving outcomes for patients with potentially curable disease (ClinicalTrials.gov identifiers: NCT02525757 and NCT02434081). The phase III PACIFIC study (ClinicalTrials.gov identifier: NCT02125461) is evaluating consolidation durvalumab vs placebo in patients with unresectable stage III NSCLC treated with definitive chemoradiation (see Table 2).

Conclusion

The elements of a successful antitumor T-cell response include generation of sufficient numbers of antitumor T cells within the lymphoid compartment; successful T-cell trafficking and extravasation out of the lymphoid compartment, through the bloodstream, and into the tumor microenvironment; and successful T-cell effector function within the tumor microenvironment, with the need to bypass immune checkpoints, soluble inhibitory factors, and inhibitory cells. Although the tumor cell can subvert each one of these steps, researchers are developing an array of strategies to overcome such subversion, also at every step. Immune checkpoint inhibition has led to long-term survival exceeding a decade in some patients with metastatic melanoma and NSCLC. Combination strategies with immune checkpoint blockade as a backbone may allow the oncology community to achieve these outstanding outcomes in greater numbers of cancer patients.

Financial Disclosure:Dr. Antonia serves on the advisory boards of Bristol-Myers Squibb (BMS), Genentech, and MedImmune/AstraZeneca (AZ). Dr. Chiappori has made a conference presentation for BMS. Dr. Creelan receives research funding from BMS and Boehringer-Ingelheim; in addition, he receives honoraria and travel grants from AZ and BMS. Dr. Soliman serves as a consultant to Celgene. The other authors have no significant financial relationship with the manufacturer of any product or provider of any service mentioned in this article.

References:

1. Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015;372:2018-28.

2. Ansell SM, Lesokhin AM, Borrello I, et al. PD-1 blockade with nivolumab in relapsed or refractory Hodgkin’s lymphoma. N Engl J Med. 2015;372:311-9.

3. Hamanishi J, Mandai M, Ikeda T, et al. Safety and antitumor activity of anti-PD-1 antibody, nivolumab, in patients with platinum-resistant ovarian cancer. J Clin Oncol. 2015 Sep 8. [Epub ahead of print]

4. Segal NH, Antonia SJ, Brahmer JR, et al. Preliminary data from a multi-arm expansion study of MEDI4736, an anti–PD-L1 antibody. J Clin Oncol. 2014;32(suppl 5S):abstr 3002.

5. Lutzky J, Antonia SJ, Blake-Haskins A, et al. A phase 1 study of MEDI4736, an anti–PD-L1 antibody, in patients with advanced solid tumors. J Clin Oncol. 2014;32(suppl 5S):abstr 3001.

6. Wolchok JD, Kluger H, Callahan MK, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013;369:122-33.

7. Topalian SL, Hodi FS, Brahmer JR, et al. Safety, activity, and immune correlates of anti–PD-1 antibody in cancer. N Engl J Med. 2012;366:2443-54.

8. Brahmer JR, Tykodi SS, Chow LQ, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366:2455-65.

9. Coley WB. The treatment of inoperable sarcoma by bacterial toxins (the mixed toxins of the Streptococcus erysipelas and the Bacillus prodigiosus). Proc R Soc Med. 1910;3:1-48.

10. Amin A, White RL, Jr. High-dose interleukin-2: is it still indicated for melanoma and RCC in an era of targeted therapies? Oncology (Williston Park). 2013;27:680-91.

11. Antony GK, Dudek AZ. Interleukin 2 in cancer therapy. Curr Med Chem. 2010;17:
3297-302.

12. Flanigan RC, Salmon SE, Blumenstein BA, et al. Nephrectomy followed by interferon alfa-2b compared with interferon alfa-2b alone for metastatic renal-cell cancer. N Engl J Med. 2001;345:1655-9.

13. Hamid O. Is IL-2 still indicated for melanoma and RCC? What a question to ask! Oncology (Williston Park). 2013;27:695,701.

14. Kantoff PW, Higano CS, Shore ND, et al. Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 2010;363:411-22.

15. Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med. 2011;364:2517-26.

16. Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711-23.

17. Robert C, Ribas A, Wolchok JD, et al. Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial. Lancet. 2014;384:1109-17.

18. Hamid O, Robert C, Daud A, et al. Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med. 2013;369:134-44.

19. Ramalingam S, Mazieres J, Planchard D, et al. Phase II study of nivolumab (anti-PD-1, BMS-936558, ONO-4538) in patients with advanced, refractory squamous non-small cell lung cancer. Int J Radiat Oncol Biol Phys. 2014;90(suppl 5s):abstr LB2.

20. Brahmer J, Reckamp KL, Baas P, et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N Engl J Med. 2015;373:123-35.

21. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;
144:646-74.

22. Finkelstein SE, Iclozan C, Bui MM, et al. Combination of external beam radiotherapy (EBRT) with intratumoral injection of dendritic cells as neo-adjuvant treatment of high-risk soft tissue sarcoma patients. Int J Radiat Oncol Biol Phys. 2012;82:924-32.

23. Gubin MM, Zhang X, Schuster H, et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature. 2014;515:577-81.

24. Rizvi NA, Hellmann MD, Snyder A, et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science. 2015;348:124-8.

25. van Rooij N, van Buuren MM, Philips D, et al. Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma. J Clin Oncol. 2013;31:e439-42.

26. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011;480:480-9.

27. Liang SC, Latchman YE, Buhlmann JE, et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur J Immunol. 2003;33:2706-16.

28. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012;12:252-64.

29. Zielinski C, Knapp S, Mascaux C, Hirsch F. Rationale for targeting the immune system through checkpoint molecule blockade in the treatment of non-small-cell lung cancer. Ann Oncol. 2013;24:1170-9.

30. McDermott D, Haanen J, Chen TT, et al. Efficacy and safety of ipilimumab in metastatic melanoma patients surviving more than 2 years following treatment in a phase III trial (MDX010-20). Ann Oncol. 2013;24:2694-8.

31. Schadendorf D, Hodi FS, Robert C, et al. Pooled analysis of long-term survival data from phase II and phase III trials of ipilimumab in unresectable or metastatic melanoma. J Clin Oncol. 2015;33:1889-94.

32. Antonia SJ, Goldberg SB, Balmanoukian AS, et al. Phase Ib study of MEDI4736, a programmed cell death ligand-1 (PD-L1) antibody, in combination with tremelimumab, a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody, in patients (pts) with advanced NSCLC. J Clin Oncol. 2015;33(suppl):abstr 3014.

33. von Pawel J, Schiller JH, Shepherd FA, et al. Topotecan versus cyclophosphamide, doxorubicin, and vincristine for the treatment of recurrent small-cell lung cancer. J Clin Oncol. 1999;17:658-67.

34. Puglisi M, Dolly S, Faria A, et al. Treatment options for small cell lung cancer-do we have more choice? Br J Cancer. 2010;102:629-38.

35. Antonia SJ, Bendell JC, Taylor MH, et al. Phase I/II study of nivolumab with or without ipilimumab for treatment of recurrent small cell lung cancer (SCLC): CA209-032. J Clin Oncol. 2015;33(suppl):abstr 7503.

36. Hammers HJ, Plimack ER, Infante JR, et al. Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC). J Clin Oncol. 2014;32(suppl 5s):abstr 4504.

37. Rizvi N, Gettinger SN, Goldman J, et al. Safety and efficacy of first-line nivolumab (NIVO: anti-programmed death-1 [PD-1]) and ipilimumab in non-small cell lung cancer (NSCLC). J Thorac Oncol. 2015;10.9 (suppl 2):abstr 786.

38. Sitkovsky MV, Kjaergaard J, Lukashev D, Ohta A. Hypoxia-adenosinergic immunosuppression: tumor protection by T regulatory cells and cancerous tissue hypoxia. Clin Cancer Res. 2008;14:5947-52.

39. Pinder MC, Rizvi NA, Goldberg SB, et al. A phase 1b open-label study to evaluate the safety and tolerability of MEDI4736, an anti-PD-L1 antibody, in combination with tremelimumab in subjects with advanced non-small cell lung cancer. J Clin Oncol. 2014;32(suppl):abstr e19137.

40. Callahan MK. A phase 1 study to evaluate the safety and tolerability of MEDI4736, an anti-programmed cell death-ligand-1 (PD-L1) antibody, in combination with tremelimumab in patients with advanced solid tumors. J Clin Oncol. 2014;33(suppl):abstr TPS3099.

41. Suntharalingam G, Perry MR, Ward S, et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med. 2006;355:1018-28.

42. Curran MA, Callahan MK, Subudhi SK, Allison JP. Response to “Ipilimumab (Yervoy) and the TGN1412 catastrophe.” Immunobiology. 2012;217:590-2.

43. Melero I, Berman DM, Aznar MA, et al. Evolving synergistic combinations of targeted immunotherapies to combat cancer. Nat Rev Cancer. 2015;15:457-72.

44. Bajor DL, Mick R, Riese MJ, et al. Combination of agonistic CD40 monoclonal antibody CP-870,893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma. Cancer Res. 2015;75(15 suppl):abstr CT137.

45. Benson DM, Jr, Bakan CE, Zhang S, et al. IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect. Blood. 2011;118:6387-91.

46. Kohrt HE, Thielens A, Marabelle A, et al. Anti-KIR antibody enhancement of anti-lymphoma activity of natural killer cells as monotherapy and in combination with anti-CD20 antibodies. Blood. 2014;123:678-86.

47. Purdy AK, Campbell KS. Natural killer cells and cancer: regulation by the killer cell Ig-like receptors (KIR). Cancer Biol Ther. 2009;8:2211-20.

48. Romagne F, Andre P, Spee P, et al. Preclinical characterization of 1-7F9, a novel human anti-KIR receptor therapeutic antibody that augments natural killer-mediated killing of tumor cells. Blood. 2009;114:2667-77.

49. Rizvi NA, Infante JR, Gibney GT, et al. A phase I study of lirilumab (BMS-986015), an anti-KIR monoclonal antibody, administered in combination with ipilimumab, an anti-CTLA4 monoclonal antibody, in patients (pts) with select advanced solid tumors. J Clin Oncol. 2013;31(suppl): abstr TPS3106.

50. Munn DH, Sharma MD, Baban B, et al. GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase. Immunity. 2005;22:633-42.

51. Mellor AL, Chandler P, Lee GK, et al. Indoleamine 2,3-dioxygenase, immunosuppression and pregnancy. J Reprod Immunol. 2002;57:143-50.

52. Munn DH, Sharma MD, Lee JR, et al. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science. 2002;297:1867-70.

53. Friberg M, Jennings R, Alsarraj M, et al. Indoleamine 2,3-dioxygenase contributes to tumor cell evasion of T cell-mediated rejection. Int J Cancer. 2002;101:151-5.

54. Liu X, Shin N, Koblish HK, et al. Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity. Blood. 2010;115:3520-30.

55. Soliman H, Mediavilla-Varela M, Antonia S. Indoleamine 2,3-dioxygenase: Is it an immune suppressor? Cancer J. 2010;16:354-9.

56. Soliman HH, Jackson E, Neuger T, et al. A first in man phase I trial of the oral immunomodulator, indoximod, combined with docetaxel in patients with metastatic solid tumors. Oncotarget. 2014;5:8136-46.

57. Holmgaard RB, Zamarin D, Munn DH, et al. Indoleamine 2,3-dioxygenase is a critical resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4. J Exp Med. 2013;210:1389-402.

58. Metz R, Rust S, Duhadaway JB, et al. IDO inhibits a tryptophan sufficiency signal that stimulates mTOR: a novel IDO effector pathway targeted by D-1-methyl-tryptophan. Oncoimmunology. 2012;1:1460-8.

59. Gibney GT, Hamid O, Gangadhar TC, et al. Preliminary results from a phase 1/2 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma. J Clin Oncol. 2014;32(suppl 5s):abstr 3010.

60. Mautino MR, Metz RA, Jaipuri F, et al. Novel specific- and dual-tryptophan-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO) inhibitors for tumor immunotherapy. Cancer Res. 2014;74(19 suppl):abstr 1633.

61. Hinrichs CS, Rosenberg SA. Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunol Rev. 2014;257:56-71.

62. Rosenberg SA, Yang JC, Sherry RM, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res. 2011;17:4550-7.

63. Robbins PF, Morgan RA, Feldman SA, et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol. 2011;29:917-24.

64. Garfall AL, Maus MV, Hwang W-T, et al. Chimeric antigen receptor T cells against CD19 for multiple myeloma. N Engl J Med. 2015;373:1040-7.

65. Beatty GL, Haas AR, Maus MV, et al. Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies. Cancer Immunol Res. 2014;2:112-20.

66. Morgan RA, Yang JC, Kitano M, et al. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther. 2010;18:843-51.

67. Maus MV, Haas AR, Beatty GL, et al. T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res. 2013;1:26-31.

68. Magee MS, Snook AE. Challenges to chimeric antigen receptor (CAR)-T cell therapy for cancer. Discov Med. 2014;18:265-71.

69. Morgan RA, Chinnasamy N, Abate-Daga D, et al. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J Immunother. 2013;36:133-51.

70. Ji RR, Chasalow SD, Wang L, et al. An immune-active tumor microenvironment favors clinical response to ipilimumab. Cancer Immunol Immunother. 2012;61:1019-31.

71. Ulloa-Montoya F, Louahed J, Dizier B, et al. Predictive gene signature in MAGE-A3 antigen-specific cancer immunotherapy. J Clin Oncol. 2013;31:2388-95.

72. Beg AA, Khan T, Antonia SJ. A new role for NFkappaB in immunosurveillance and its implications for cancer immunotherapy. Oncoimmunology. 2013;2:e25963.

73. Tchekmedyian N, Zheng H, Beg AA, et al. Preclinical rationale for a phase I/II study of pembrolizumab (P) and vorinostat (V) in immune therapy naive and pretreated stage IV NSCLC. J Thorac Oncol. 2015;10.9(suppl 2): abstr 734.

74. Juergens RA, Wrangle J, Vendetti FP, et al. Combination epigenetic therapy has efficacy in patients with refractory advanced non-small cell lung cancer. Cancer Discov. 2011;1:598-607.

75. Wrangle J. Epigenetic therapy and sensitization of lung cancer to immunotherapy. Cancer Res. 2013;73(8 suppl):abstr 4619.

76. Hellmann MD, Garon EB, Gandhi L, et al. Efficacy of pembrolizumab in key subgroups of patients with advanced NSCLC. J Thorac Oncol. 2015;10.9(suppl 2:):abstr 3057.

77. Garon EB, Wolf B, Lisberg A, et al. Prior TKI therapy in NSCLC EGFR mutant patients associates with lack of response to anti-PD-1 treatment. J Thorac Oncol. 2015;10.9(suppl 2): abstr 2172.

78. Gettinger S, Rizvi N, Chow LQ, et al. Nivolumab (anti-PD-1; BMS-936558, ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) or erlotinib (ERL) in advanced non-small cell lung cancer (NSCLC). Ann Oncol. 2014;25(suppl 4):iv361-iv372.

79. Janne PA, Yang JC, Kim DW, et al. AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer. N Engl J Med. 2015;372:1689-99.

80. Sequist LV, Soria JC, Goldman JW, et al. Rociletinib in EGFR-mutated non-small-cell lung cancer. N Engl J Med. 2015;372:1700-9.

81. Kitamura M, Connolly A. AstraZeneca temporarily halts cancer drug combination trials. Bloomberg Business. October 9, 2015. http://www.bloomberg.com/news/articles/2015-10-09/astrazeneca-temporarily-halts-cancer-drug-combination-studies.

82. Vanneman M, Dranoff G. Combining immunotherapy and targeted therapies in cancer treatment. Nat Rev Cancer. 2012;12:237-51.

83. Hodi FS, Lawrence D, Lezcano C, et al. Bevacizumab plus ipilimumab in patients with metastatic melanoma. Cancer Immunol Res. 2014;2:632-42.

84. Hu-Lieskovan S, Robert L, Homet Moreno B, Ribas A. Combining targeted therapy with immunotherapy in BRAF-mutant melanoma: promise and challenges. J Clin Oncol. 2014;32:2248-54.

85. Ribas A, Hodi FS, Callahan M, et al. Hepatotoxicity with combination of vemurafenib and ipilimumab. N Engl J Med. 2013;368:1365-6.

86. Harding JJ, Pulitzer M, Chapman PB. Vemurafenib sensitivity skin reaction after ipilimumab. N Engl J Med. 2012;366:866-8.

87. Johnson DB, Wallender EK, Cohen DN, et al. Severe cutaneous and neurologic toxicity in melanoma patients during vemurafenib administration following anti-PD-1 therapy. Cancer Immunol Res. 2013;1:373-7.

88. Ribas A, Butler M, Lutzky J, et al. Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) and/or MEK (trametinib) inhibitors in advanced melanoma. J Clin Oncol. 2015;33(suppl):abstr 3003.

89. Wu YL, Park K, Soo RA, et al. INSPIRE: a phase III study of the BLP25 liposome vaccine (L-BLP25) in Asian patients with unresectable stage III non-small cell lung cancer. BMC Cancer. 2011;11:430.

90. Vansteenkiste J, Zielinski M, Linder A, et al. Adjuvant MAGE-A3 immunotherapy in resected non-small-cell lung cancer: phase II randomized study results. J Clin Oncol. 2013;31:2396-403.

91. Quoix E, Ramlau R, Westeel V, et al. Therapeutic vaccination with TG4010 and first-line chemotherapy in advanced non-small-cell lung cancer: a controlled phase 2B trial. Lancet Oncol. 2011;12:1125-33.

92. Neninger Vinageras E, de la Torre A, Osorio Rodríguez M, et al. Phase II randomized controlled trial of an epidermal growth factor vaccine in advanced non-small-cell lung cancer. J Clin Oncol. 2008;26:1452-8.

93. Nemunaitis J, Dillman RO, Schwarzenberger PO, et al. Phase II study of belagenpumatucel-L, a transforming growth factor beta-2 antisense gene-modified allogeneic tumor cell vaccine in non-small-cell lung cancer. J Clin Oncol. 2006;24:4721-30.

94. Dessureault S, Noyes D, Lee D, et al. A phase-I trial using a universal GM-CSF-producing and CD40L-expressing bystander cell line (GM.CD40L) in the formulation of autologous tumor cell-based vaccines for cancer patients with stage IV disease. Ann Surg Oncol. 2007;14:869-84.

95. Creelan BC, Antonia S, Noyes D, et al. Phase II trial of a GM-CSF-producing and CD40L-expressing bystander cell line combined with an allogeneic tumor cell-based vaccine for refractory lung adenocarcinoma. J Immunother. 013;36:442-50.

96. Butts C, Socinski MA, Mitchell PL, et al. Tecemotide (L-BLP25) versus placebo after chemoradiotherapy for stage III non-small-cell lung cancer (START): a randomised, double-blind, phase 3 trial. Lancet Oncol. 2014;15:59-68.

97. Zippelius A, Batard P, Rubio-Godoy V, et al. Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance. Cancer Res. 2004;64:2865-73.

98. Nielsen MB, Marincola FM. Melanoma vaccines: the paradox of T cell activation without clinical response. Cancer Chemother Pharmacol. 2000;46(suppl):S62-S66.

99. Weber JS, Kudchadkar RR, Yu B, et al. Safety, efficacy, and biomarkers of nivolumab with vaccine in ipilimumab-refractory or -naive melanoma. J Clin Oncol. 2013;31:4311-8.

100. Hodi FS, Butler M, Oble DA, et al. Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients. Proc Natl Acad Sci USA. 2008;105:3005-10.

101. Hodi FS, Mihm MC, Soiffer RJ, et al. Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients. Proc Natl Acad Sci USA. 2003;100:4712-7.

102. Hodi FS, Lee S, McDermott DF, et al. Ipilimumab plus sargramostim vs ipilimumab alone for treatment of metastatic melanoma: a randomized clinical trial. JAMA. 2014;312:1744-53.

103. Antonia SJ, Larkin J, Ascierto PA. Immuno-oncology combinations: a review of clinical experience and future prospects. Clin Cancer Res. 2014;20:6258-68.

104. Lynch TJ, Bondarenko I, Luft A, et al. Ipilimumab in combination with paclitaxel and carboplatin as first-line treatment in stage IIIB/IV non-small-cell lung cancer: results from a randomized, double-blind, multicenter phase II study. J Clin Oncol. 2012;30:2046-54.

105. Reck M, Bondarenko I, Luft A, et al. Ipilimumab in combination with paclitaxel and carboplatin as first-line therapy in extensive-disease-small-cell lung cancer: results from a randomized, double-blind, multicenter phase 2 trial. Ann Oncol. 2013;24:75-83.

106. Antonia SJ, Brahmer JR, Gettinger S, et al. Nivolumab (Anti-PD-1; BMS-936558, ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC). Int J Radiat Oncol Biol Phys. 2014;90:S2.

107. Camidge R, Liu SV, Powderly J, et al. Atezolizumab (MPDL3280A) combined with platinum-based chemotherapy in non–small cell lung cancer (NSCLC): a phase Ib safety and efficacy update. J Thorac Oncol. 2015;10:s176-s177.

108. Burnette B, Weichselbaum RR. Radiation as an immune modulator. Semin Radiat Oncol. 2013;23:273-80.

109. Reits EA, Hodge JW, Herberts CA, et al. Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy. J Exp Med. 2006;203:1259-71.

110. Lee Y, Auh SL, Wang Y, et al. Therapeutic effects of ablative radiation on local tumor require CD8+ T cells: changing strategies for cancer treatment. Blood. 2009;114:589-95.

111. Sharabi AB, Nirschl CJ, Kochel CM, et al. Stereotactic radiation therapy augments antigen-specific PD-1-mediated antitumor immune responses via cross-presentation of tumor antigen. Cancer Immunol Res. 2015;3:345-55.

112. Deng L, Liang H, Burnette B, et al. Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. J Clin Invest. 2014;124:687-95.


113. Zeng J, See AP, Phallen J, et al. Anti-PD-1 blockade and stereotactic radiation produce long-term survival in mice with intracranial gliomas. Int J Radiat Oncol Biol Phys. 2013;86:343-9.

114. Twyman-Saint Victor C, Rech AJ, Maity A, et al. Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature. 2015;520:373-7.

115. Postow MA, Callahan MK, Barker CA, et al. Immunologic correlates of the abscopal effect in a patient with melanoma. N Engl J Med. 2012;366:925-31.

116. Grimaldi AM, Simeone E, Giannarelli D, et al. Abscopal effects of radiotherapy on advanced melanoma patients who progressed after ipilimumab immunotherapy. Oncoimmunology. 2014;3:e28780.