CTLA-4 Antibodies: New Directions, New Combinations

Checkpoint blockade is a transformative therapeutic approach to a broad spectrum of malignancies because it increases the power of antitumor immunity to obtain durable responses. Cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) is the prototypical inhibitory checkpoint receptor. Since US Food and Drug Administration approval of the anti–CTLA-4 antibody ipilimumab for use in patients with melanoma, there has been ever-increasing excitement among oncologists about new ways to use this method of releasing the “brakes” on patients’ endogenous immune systems. This review will summarize the preclinical and clinical development of CTLA-4–blocking antibodies, discuss recent insights into the biology of CTLA-4 blockade, review the use of these antibodies in combination with established and novel therapeutic modalities, and comment on ongoing questions regarding their administration.

Introduction

The journey from bench to bedside

Cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4; also known as CD152) is expressed on the surface of T cells, where it primarily suppresses their early stages of activation by inducing inhibitory downstream T-cell receptor (TCR) signaling and counteracting activity of the T-cell costimulatory receptor, CD28.[1,2] CTLA-4 is thought to outcompete CD28 for B7 ligands (CD80 and CD86) on the surface of antigen-presenting cells by binding them with higher affinity and avidity.[3] In preclinical studies, blockade of CTLA-4 led to a 1.5-fold to 2-fold increase in T-cell proliferation and a 6-fold increase in interleukin-2 production.[4]

The physiologic role of CTLA-4 is not only to suppress effector T cells (Teffs), but also to increase the function of immunosuppressive CD4+FoxP3+ regulatory T cells (Tregs). Treg-specific CTLA-4 deficiency was shown to diminish the suppressive capacity of Tregs in cell culture, resulting in upregulation of CD80 and CD86 expression on dendritic cells (DCs).[5] CTLA-4 blockade has been shown to promote T-cell activation, and in preclinical models, to deplete intratumoral Tregs in a process dependent on the presence of Fcγ receptor-expressing macrophages within the tumor microenvironment.[6,7]

CTLA-4 was shown to play a critical role in maintaining immunologic homeostasis when mice genetically deficient in CTLA-4 developed a rapidly progressive, fatal lymphoproliferative disease, characterized by multiorgan T-cell infiltration and death by 3 to 4 weeks of age.[8,9] However, Leach et al subsequently demonstrated in a mouse model that blockade of CTLA-4 with antibodies did not cause lethal systemic autoimmunity.[10] Moreover, anti–CTLA-4 treatment in this preclinical study not only resulted in rejection of pre-established tumors but also in immunity to a secondary exposure to tumor cells without additional CTLA-4 blockade, thereby establishing the development of immune memory.

Based on these preclinical findings, clinical testing of two antibodies that block CTLA-4 in humans, ipilimumab and tremelimumab, was begun. Ipilimumab belongs to the immunoglobulin G (IgG) 1 class of fully human monoclonal antibodies (mAbs) and has a half-life of 12 to 14 days. Tremelimumab belongs to the IgG2 class, which causes less antibody-dependent cellular toxicity than IgG1, and has a half-life of 22 days. Lessons learned from the initial phase I/II studies, which have been summarized in prior reviews, include the emergence of a unique toxicity profile, composed of immune-related adverse events (irAEs), and new response patterns in which new lesions are viewed as part of the total tumor burden and not regarded immediately as progressive disease.[11,12]

US Food and Drug Administration (FDA) approval of ipilimumab was ultimately based on the results of a randomized phase III trial for patients with previously treated, unresectable stage III or IV melanoma who received ipilimumab 3 mg/kg with or without glycoprotein (gp)100 peptide vaccine vs gp100 peptide vaccine alone.[13] Median overall survival (OS) in the ipilimumab and ipilimumab + gp100 cohorts was 10.1 and 10.0 months, respectively, vs 6.4 months for the gp100 control arm (hazard ratio [HR], 0.68; P < .001). More importantly, ipilimumab had an effect on long-term survival, with 18% of the ipilimumab-treated patients surviving beyond 2 years compared with 5% of patients who received the gp100 peptide vaccine alone.

Tremelimumab was tested in a randomized phase III trial in patients with advanced melanoma who received either 15 mg/kg every 3 months as a single agent or dacarbazine/temozolomide.[14] The endpoint of improved OS was not reached despite a proportion of subjects experiencing a durable response after treatment with tremelimumab. The lack of an OS benefit may have been due to crossover to an expanded-access ipilimumab program by patients who received chemotherapy in the control arm. Further, pharmacokinetic simulations subsequently indicated that, despite tremelimumab’s longer half-life, a 15-mg/kg every-3-month dosing strategy resulted in only 50% of subjects reaching the drug’s target concentration.[15] Approximately 90% of subjects reached target levels when treated with tremelimumab 10 mg/kg every 4 weeks for 6 months. Tremelimumab recently showed encouraging clinical activity with the 15-mg/kg every-3-month dosing strategy in previously treated patients with advanced malignant mesothelioma in a phase II trial.[16] For these reasons, tremelimumab continues to be investigated at different doses and in a variety of malignancies and combination regimens, which will be discussed later in this article.

Back to the bench: next-generation sequencing and further insights into the biology of CTLA-4 blockade

Only a subset of patients and tumor types benefits from CTLA-4 blockade. Therefore, immense effort has been expended to understand tumor and host characteristics that contribute to response. Next-generation sequencing may prove to be a valuable tool in helping to achieve this goal.

Approximately 40% of cutaneous metastatic melanomas have an activating mutation that results in the substitution of glutamic acid for valine at codon 600 (BRAF V600E) and leads to constitutive activation of downstream signaling through the mitogen-activated protein kinase (MAPK) pathway. [17,18] Although this mutation is not thought to correlate with response to anti–CTLA-4 therapy,[19] recent data suggest that a mutation in RAS (rat sarcoma) may correlate with response to ipilimumab.[20] In this retrospective study, patients with metastatic melanoma who harbored a mutation in NRAS (neuroblastoma RAS) had a clinical benefit rate of 41% from ipilimumab therapy vs 22% for wild-type patients (P = .018; N = 137).

Further, a preclinical study found that phosphatase and tensin homolog (PTEN) represses the expression of immunosuppressive cytokines by blocking the phosphatidylinositol 3-kinase (PI3K) pathway, which is a downstream target of RAS.[21] Indeed, PTEN loss in malignant melanoma samples was associated with a host response that was not brisk, which could, in theory, predict a poor response to CTLA-4 blockade. The immunologic consequences of these signaling pathways are an area of active research; whether they influence immunotherapy treatment outcomes requires additional investigation.

Another hypothesis implicates the role of immunogenic neoantigens. Different types of malignancies vary with respect to the number of cancer-causing mutations that may encode proteins foreign to the immune system and directly correlate with response to T cell–based immunotherapy.[22,23] However, there does seem to be checkpoint inhibitor activity in tumors with lower median mutational loads, and there also appears to be a lack of activity in certain subjects with tumors classically associated with a high mutational burden. This discrepancy may be due to interindividual differences in the mutational load across a given tumor type, or there may be specific mutations that are more likely to promote an immune response.

To help shed light on this issue, two recent studies have taken a closer look at cancer genome data to determine whether there are mutations that contribute to a T-cell response. The first used bioinformatics and in vitro strategies in a patient with stage IV melanoma to show that a peptide resulting from a mutation in the ATR (ataxia telangiectasia and Rad3 related) gene generated specific T-cell reactivity that increased strongly after successful treatment with ipilimumab.[24] The second performed whole-exome sequencing of tumor DNA from 11 patients who had long-term benefit and 14 who had minimal or no benefit from ipilimumab treatment for advanced melanoma.[25] A preliminary association between certain neoantigens and clinical outcomes was seen.

Whole-exome sequencing and TCR quantitative sequencing have recently been applied to identify patient germline and immune characteristics that may predict clinical benefit from CTLA-4 blockade. In patients with metastatic melanoma treated with ipilimumab, whole-exome sequencing of germline DNA from 30 objective responders and 30 nonresponders identified several single nucleotide polymorphisms that cosegregated with clinical outcomes.[26] Although these results need to be confirmed in functional and larger prospective studies, the genes identified represent chemokine receptors and thus support the biologic plausibility of this result. The effects of anti–CTLA-4 therapy on the T-cell repertoire were recently studied using next-generation sequencing of the TCRβ gene from T cells isolated from samples of peripheral blood mononuclear cells; 25 metastatic castration-resistant prostate cancer (CRPC) patients treated with ipilimumab and granulocyte macrophage colony-stimulating factor (GM-CSF), 21 metastatic melanoma patients treated with tremelimumab, and 9 untreated healthy control subjects were included.[27] Although blockade of CTLA-4 caused global turnover of the T-cell repertoire and an increase in TCR diversity, improved OS was associated only with maintenance of high-frequency TCR clonotypes throughout treatment. This result suggests that high-avidity, pre-existing T cells may be important to the antitumor response seen with CTLA-4 blockade.

These advances also suggest that a more personalized strategy may ultimately be feasible for the application of CTLA-4 blockade. If expeditious and reproducible prescreening methods could be developed, those patients and tumor types most likely to benefit could be identified. Further, these techniques give important mechanistic insights that could aid in the design of combination approaches for the treatment of patients who are unlikely to respond to anti–CTLA-4 monotherapy.

Novel Combinatorial Strategies

To improve on the number of patients who benefit from CTLA-4–blocking antibody therapy, preclinical and clinical studies have investigated combining CTLA-4 blockade with a variety of other approaches. The major mechanistic principles behind such combinations are exploitation of cytotoxic and tumor-specific immune effector cells, induction of immunogenic cell death,[28] alleviation of tumor-associated immune suppression, and counterbalance of immune escape mechanisms (Figure).

Chemotherapy and antiangiogenic therapy

Conventional chemotherapy is an effective treatment modality for a variety of advanced cancers. This efficacy may derive, at least in part, from the capacity of certain chemotherapeutic agents to stimulate the immune system.[29] For example, anthracyclines can trigger immunogenic cell death,[30] and paclitaxel may facilitate immunologic priming by binding to toll-like receptors on the surface of immature DCs, facilitating maturation and maximizing the extent of DC activation.[31] Cyclophosphamide depletes Tregs,[32] while fluorouracil and gemcitabine selectively eliminate myeloid-derived suppressor cells (MDSCs).[33,34] MDSCs are a heterogeneous population of immunosuppressive cells that have been associated with OS in patients with melanoma.[35,36]

Although these immunomodulatory effects of chemotherapy suggest a potential for synergy with CTLA-4 blockade, this has yet to be proven in patients with advanced melanoma. A first-line trial reported improved OS when comparing dacarbazine and ipilimumab 10 mg/kg vs dacarbazine alone (11 vs 9 months, respectively); however, there was no ipilimumab monotherapy arm to prove that there was indeed synergy from the combination.[37] A phase I study compared ipilimumab 10 mg/kg alone (I group) or in combination with dacarbazine (D group) or carboplatin/paclitaxel (CP group) in patients with previously untreated advanced melanoma.[38] Although no major pharmacokinetic or pharmacodynamic interactions were observed when ipilimumab was administered with chemotherapy, response rates were 29.4% (I group), 27.8% (D group), and 11.1% (CP group). At present, National Comprehensive Cancer Network (NCCN) member institutions do not recommend the combination of ipilimumab and dacarbazine in advanced melanoma because of liver toxicity and unproven clinical benefit over ipilimumab alone.[37] In addition, given that melanoma response rates to chemotherapy are typically low,[39] chemotherapy may not be the best combinatorial partner for CTLA-4 blockade.

The combination of chemotherapy and CTLA-4 blockade has shown an acceptable safety and tolerability profile, as well as promising clinical activity in phase II trials for both extensive-disease–small-cell lung cancer (ED-SCLC) and non–small-cell lung cancer (NSCLC).[40,41] Both trials had a similar design, in which chemotherapy-naive patients received concurrent ipilimumab (ipilimumab + paclitaxel/carboplatin followed by placebo + paclitaxel/carboplatin) or phased ipilimumab (placebo + paclitaxel/carboplatin followed by ipilimumab + paclitaxel/carboplatin). The ED-SCLC trial reported improved “immune-related” progression-free survival (PFS) with the phased regimen (HR, 0.64; P = .03) compared with the concurrent regimen (HR, 0.75; P = .11), as did the NSCLC trial (HR, 0.72; 95% confidence interval [CI], 0.50–1.06; P = .05 vs HR, 0.81; CI, 0.55–1.17; P = .13). The NSCLC trial also noted improved efficacy for squamous histology, which is reported to have a greater abundance of tumor-infiltrating lymphocytes.[42] The efficacy of CTLA-4 blockade in these diseases continues to be tested in several actively recruiting clinical trials (NCT01331525, NCT01454102, NCT02039674). There are also ongoing clinical studies investigating ipilimumab in combination with gemcitabine for recurrent pancreatic cancer (NCT01473940), in combination with gemcitabine and cisplatin for metastatic urothelial carcinoma (NCT01524991), and in combination with paclitaxel and carboplatin for squamous NSCLC (NCT01285609).

Bevacizumab is a humanized mAb against vascular endothelial growth factor (VEGF) and is commonly used in combination with chemotherapy for a variety of malignancies. In melanoma, a randomized phase II study in patients with previously untreated metastatic melanoma compared carboplatin and paclitaxel with and without bevacizumab.[43] Although this trial did not reach its primary objective of statistically significant improvement in PFS, exploring antiangiogenic approaches in patients with melanoma is still of interest because VEGF is a potent inhibitor of DC maturation and T-cell responses.[44,45] VEGF levels were associated with clinical response and OS in advanced melanoma patients treated with ipilimumab.[46] A recent phase I study of bevacizumab + ipilimumab in 46 metastatic melanoma patients showed clinical responses, with manageable toxicity.[47] There were 8 patients with partial responses, 22 patients with stable disease, and a 67.4% disease control rate. This clinical activity was preliminary but nevertheless favorable when compared with ipilimumab alone, which had a 28.5% disease control rate in the trial that led to its approval.[13] Combined VEGF and CTLA-4 blockade seemed to increase immune cell infiltration into the tumor and circulating memory T cells in the peripheral blood. This combination is currently being evaluated in a phase II randomized study (NCT01950390).

Non–antigen-specific vaccination

Localized tumor-destructive techniques may also have the capacity to cause antigen release and to affect immunosuppressive elements, which in aggregate may be beneficial for combination strategies with anti–CTLA-4 therapy. For example, palliative radiotherapy in a patient with metastatic melanoma caused disease regression outside the irradiated field in areas that had been worsening on ipilimumab alone.[48] In-depth immune monitoring of the patient revealed enhanced humoral immunity to several antigens, as well as a decrease in the MDSC population. A retrospective analysis of 29 patients who received extracranial radiation during ipilimumab therapy revealed no significant increase in AEs related to ipilimumab nor any compromise in its efficacy.[49] These clinical cases, in combination with supportive preclinical data, have led to multiple prospective trials investigating radiotherapy with ipilimumab for patients with melanoma (NCT01703507, NCT01497808, NCT01565837, NCT01689974), cryoablation with ipilimumab for patients with breast cancer (NCT01502592), and transarterial chemoembolization or radiofrequency ablation with tremelimumab for patients with hepatocellular carcinoma (NCT01853618). Despite encouraging preclinical and phase II results in prostate cancer,[50,51] a recent phase III trial comparing ipilimumab with placebo after radiotherapy in patients with metastatic CRPC with disease progression after docetaxel failed to show a significant OS benefit.[52] However, anti–CTLA-4 therapy did improve PFS and, in a subgroup analysis, seemed to improve survival in patients without visceral metastasis. There are many theories about why this was not a positive study, one of which postulates that visceral metastases may lead to an immunosuppressive cytokine profile, which cannot be overcome by CTLA-4 blockade. An ongoing phase III trial is comparing the efficacy of ipilimumab vs placebo in asymptomatic or minimally symptomatic patients with metastatic chemotherapy-naive CRPC (NCT01057810). Finally, viruses are a new technique to induce tumor antigen release and subsequent development of tumor-specific immunity (see sidebar, “Oncolytic Virotherapy”).

Antigen-specific and cytokine-secreting vaccination strategies

In addition to ablative techniques to induce in vivo tumor antigen release and cross-presentation as discussed above, antigen-specific immunotherapeutic strategies are another approach. Several tumor-associated antigens have been identified in melanoma, including, but not limited to, MAGE, Melan-A (MART-1), gp100, tyrosinase, and the cancer-testis antigen NY-ESO-1. While it is attractive to believe that combination of a vaccine and CTLA-4 blockade would potentiate a robust antitumor response, the failure of the monovalent gp100 vaccine to have any impact in the phase III trial that led to FDA approval of ipilimumab for metastatic melanoma highlights the significant challenges to development of an efficacious tumor vaccine.[13] Ongoing trials are currently focusing on testing multivalent vaccines and developing novel techniques of enhancing antigen presentation.[53,54]

An emerging strategy has been to vaccinate with irradiated, autologous tumor cells engineered to secrete GM-CSF (GVAX). In one study, ipilimumab 10 mg/kg was given alone and in combination with GVAX to a total of 30 patients with metastatic pancreatic ductal carcinoma.[55] The median OS (3.6 vs 5.7 months; HR, 0.51; P = .072) and 1-year OS (7% vs 27%) favored the combination over ipilimumab alone. There was an enhancement in the T-cell repertoire among patients with an OS > 4.3 months, which may support the theory that GM-CSF production at the site of the vaccine attracts host antigen-presenting cells and enhances their function in vivo.[56] While not a vaccine approach, GM-CSF by itself was added to ipilimumab in patients with metastatic melanoma and improved OS and decreased high-grade AEs compared with ipilimumab alone in a randomized study.[57]

Targeted therapy

Compared with CTLA-4 blockade, for patients with BRAF-mutant melanoma, BRAF inhibitors (BRAFis) such as vemurafenib and dabrafenib cause a more rapid response. However, the median PFS is only 6 months for monotherapy, and 9.4 months when dabrafenib is combined with trametinib, a selective MEK inhibitor.[39,58,59] In addition to blocking biochemical pathways necessary for tumor cell survival, these and other targeted therapies appear to increase antigen expression, improve lymphocyte trafficking, and promote an inflammatory tumor microenvironment.[60] Although resistance usually develops owing to compensatory changes or secondary mutations within the targeted pathway, these immunomodulatory properties raise the possibility that combination or sequencing with CTLA-4 blockade may convert the initially rapid, yet usually transient, tumor regression into longer-lasting benefit.

Despite encouraging preclinical data, additional clinical investigation is necessary to fully understand the possibility of combining targeted therapy with immunotherapy. A phase I trial combining vemurafenib and ipilimumab was stopped early because of hepatotoxicity.[61] Similarly, a study of combination dabrafenib, trametinib, and ipilimumab was also stopped early due to gastrointestinal AEs, but it is possible that dabrafenib may be tolerable with ipilimumab.[62] In a recent retrospective analysis, prior treatment with ipilimumab and interleukin-2 did not appear to negatively influence response to BRAFi in some patients, while outcomes with ipilimumab following BRAFi discontinuation were poor.[63] Ackerman et al postulate that those patients who progress on a BRAFi may have poor performance status and insufficient lifespan to benefit from ipilimumab, which typically requires weeks or months to show response.[63] A decrease in melanoma antigen expression and CD8+ T-cell infiltration has also been noted in tumor biopsy specimens obtained at the time of progression from patients on a BRAFi.[64] Alternatively, resistance mutations in the MAPK or PI3-Akt pathway after BRAFi therapy may lead to repression of antitumor immunity. Nevertheless, the potential for confounding was high in this retrospective analysis, and the general consensus among practitioners is to use vemurafenib or dabrafenib before CTLA-4 therapy in patients with BRAF-mutated tumors who are symptomatic or have high tumor burden, given the greater likelihood of a rapid response. However, patients should be enrolled in a clinical protocol whenever possible, as the field eagerly awaits the results of randomized clinical trials (NCT01673854, NCT01940809, NCT01767454) to help guide treatment.

Combinations of T-cell immunomodulatory antibodies

As reviewed by Pardoll[65] and Ribas,[66] CTLA-4 serves to regulate early T-cell activation, while programmed cell death 1 (PD-1) signaling primarily functions to limit T-cell activity within peripheral tissues. The therapeutic implications of targeting the PD-1 pathway are discussed in depth in the article by Drs. Kim and Eder on page 15 of this issue. Some studies have investigated combinations of anti–PD-1 agents with ipilimumab. A phase I study of nivolumab and ipilimumab had a high response rate, which was associated with favorable OS compared with historical data.[67,68] Specifically, long-term follow-up revealed that ≥ 80% tumor reduction was observed in 42% of the initial 53 patients with advanced melanoma, with a 2-year survival rate of 79%. The median OS was 39.7 months across all the concurrent regimens, while the median OS for patients who received nivolumab after progression on ipilimumab was 13 months. The maximum tolerated doses were nivolumab, 1 mg/kg, and ipilimumab, 3 mg/kg. Phase II and III trials investigating the concurrent combination of nivolumab and ipilimumab vs either agent alone in patients with advanced melanoma have completed enrollment (NCT01844505, NCT01927419), and the results are eagerly awaited. Finally, phase I trials have also shown promising activity of ipilimumab and nivolumab combination therapy in other diseases; in metastatic renal cell carcinoma, the combination had an impressive 46% objective response rate.[69] In the future, it is possible that a biomarker-driven approach will also play a role in how these immunotherapeutic strategies are combined or sequenced to maximize clinical benefit.

While immune checkpoint receptors such as CTLA-4 and PD-1 deliver inhibitory signals to the T cell, there is growing interest in targeting costimulatory receptors that enhance T-cell function. As an example, 4-1BB signaling delivers a dual mitogenic signal for T-cell activation and growth, and administration of anti–4-1BB mAbs eradicated large tumors in mice.[70] BMS-663513 is a fully humanized mAb agonist of 4-1BB tested in phase I clinical trials, initially only in melanoma patients but expanded to renal cell carcinoma and ovarian cancer patients.[71] Clinical activity represented by partial responses and sustained stable disease was observed in the 1, 3, and 10 mg/kg groups, but there was more toxicity at doses > 6 mg/kg. PF-05082566 is a fully humanized IgG2 agonist mAb that also targets 4-1BB. A recent first-in-human phase I study in 34 patients with a variety of malignancies reported that the drug was well tolerated at 2.4 mg/kg, with no adverse events ≥ grade 2.[72] There was also preliminary single-agent activity in 24 evaluable patients, reflected by 1 patient with a partial response at the 0.6 mg/kg dose, 1 patient with a mixed response at the 0.06 mg/kg dose, and 7 patients with stable disease across multiple doses. There have been no human studies combining PF-05082566 with anti–CTLA-4 blockade, but there is promising preclinical data in colon cancer and glioma mouse models showing that 4-1BB agonism may abrogate the toxicity of CTLA-4 blockade.[73,74] As summarized in a review by Sugamura et al, OX40 is another costimulatory receptor on T cells, with preclinical data supporting amplification of antitumor immunity against various tumor types, such as breast, melanoma, and prostate.[75] A phase I clinical trial using a mouse mAb that agonizes human OX40 in advanced cancer patients reported an acceptable toxicity profile and regression of at least one metastatic lesion in 12 of 30 patients.[76] Combining an agonist anti-OX40 mAb with CTLA-4 blockade boosted antitumor immunity in a preclinical model.[77] The combination of immune checkpoint inhibitors with costimulatory agonists may be a viable strategy, particularly against poorly immunogenic tumors.

Novel methods for combating immune resistance

The precise immunologic factors that contribute to immunotherapy resistance remain unknown, but a number of studies have investigated Treg, Teff and other immune-inhibitory molecules in the tumor microenvironment.[78,79] The cytosolic enzyme indoleamine 2,3-dioxygenase (IDO) is expressed by both tumor cells and infiltrating myeloid cells and produces bioactive tryptophan metabolites that appear to exert suppressive activity on T cells.[80] Preclinical work revealed that CTLA-4 blockade may be synergistic with IDO inhibition to enhance the infiltration of tumor-specific Teffs and lead to the regression of both IDO-expressing and IDO-nonexpressing poorly immunogenic tumors.[81] A recent phase I/II study administered ipilimumab with INCB024360, a selective IDO1 inhibitor, to patients with metastatic melanoma. The combination was generally well tolerated and showed disease control in 75% (6/8) at the 25 mg bid dose.[82] Finally, IDO appears to be unregulated by transforming growth factor β (TGF-β) signaling, which leads to Treg infiltration and an immunosuppressive tumor microenvironment.[83] Indeed, an anti–CTLA-4 mAB given in combination with a type I TGF-β receptor kinase inhibitor suppressed tumor growth and metastasis in a transgenic melanoma model that was correlated with suppression of IDO activity and an increase in the Teff-to-Treg ratio.[84]

In addition to modulation of the T-cell response, other immunologic cell populations are being targeted as a possible mechanism for overcoming immune resistance. For example, natural killer cells are cytotoxic in a manner similar to that of Teffs, but without using a TCR for recognition. Instead, they express killer immunoglobulin-like receptors (KIRs), which bind human leukocyte antigen (HLA) class I molecules on target cells, thereby delivering an inhibitory signal that prevents natural killer cell–mediated cytotoxicity.[85] Anti-KIR antibodies may release these inhibitory KIR-mediated signals, thereby enabling tumor cytotoxicity and immune clearance. A phase I trial is currently investigating lirilumab (an anti-KIR mAb) in combination with ipilimumab in patients with NSCLC, metastatic melanoma, and CRPC (NCT01750580).

Ongoing Questions

Dosing

Although the FDA and the European Medicines Agency have approved ipilimumab at a dose of 3 mg/kg, it is not clear that this is always the optimal dose. A double-blind phase II study in patients with advanced melanoma defined a dose-response relationship by comparing ipilimumab at 0.3, 3, and 10 mg/kg every 3 weeks, followed by maintenance doses every 12 weeks.[86] The 10 mg/kg cohort had the greatest response rate (11.1%), followed by 3 mg/kg (4.2%) and 0.3 mg/kg (0%). The rate of grade 3/4 irAEs was also higher with increased ipilimumab dose (25% vs 7% vs 0%). We await the results of a phase III randomized trial comparing 10 mg/kg vs 3 mg/kg (NCT01515189). Dosing of ipilimumab in various combination strategies may be important, as many doses, including 1, 3, and 10 mg/kg, have been explored in combination approaches. Finally, recent data on the combination of ipilimumab and nivolumab in a first-line NSCLC cohort showed toxicity with the 3 + 1 mg/kg (ipilimumab and nivolumab, respectively) and 1 + 3 mg/kg (ipilimumab and nivolumab, respectively) dosing that was more severe than in melanoma and renal cell carcinoma trials. It necessitated the addition of a 1 + 1 mg/kg cohort.[87] It is possible that different doses of immunotherapy combinations may be appropriate for patients with different underlying tumors.

Schedule

The relative benefit of re-induction and maintenance therapy with anti–CTLA-4 agents is also uncertain. In the ipilimumab + gp100 trial, a select group of patients who initially benefited from ipilimumab and then subsequently progressed were eligible for ipilimumab re-induction.[13] Of the 31 patients given re-induction, 21 achieved a partial or complete response or stable disease. Based on these data, NCCN guidelines state that re-induction with ipilimumab may be considered for patients who experienced no significant systemic toxicity and who relapsed after an initial clinical response or who progress after stable disease for > 3 months.[88] This strategy’s efficacy will be prospectively evaluated in a trial comparing re-induction vs physician’s choice of chemotherapy (NCT00495066). Unfortunately, this and many of the previously mentioned accruing trials do not address the question of whether there is a clinical benefit from ipilimumab maintenance therapy. Prospective randomized trials are needed to help determine whether patients can demonstrate lasting antitumor immunity after a defined treatment course of anti–CTLA-4 therapy or whether continued dosing confers additional clinical benefit.

Treatment setting

The success of CTLA-4 blockade for metastatic melanoma has led to investigation into the efficacy of its use earlier in the disease course. Ipilimumab is being studied in two large adjuvant studies for patients with stage III melanoma. The first is investigating ipilimumab vs interferon and closed to accrual in August 2014 (Eastern Cooperative Oncology Group 1609, NCT01274338). The second is investigating ipilimumab vs observation (European Organisation for Research and Treatment of Cancer 18071, NCT00636168). The first results of ipilimumab in the adjuvant setting were recently presented; they demonstrated a median relapse-free survival of 26.1 months for patients treated with ipilimumab at 10 mg/kg vs 17.1 months for those receiving placebo. Of note, approximately 50% of the ipilimumab-treated patients discontinued the drug due to treatment-related AEs, and there were five treatment-related deaths (1.1%) in the ipilimumab group (three from colitis and one each from myocarditis and Guillain-Barré syndrome). The rates of irAEs in the adjuvant setting appear grossly similar to those of 10 mg/kg of ipilimumab in the metastatic setting, although endocrinopathies may be more frequent. The OS data from this trial, additional data for 3 mg/kg ipilimumab, and direct comparison with interferon are needed to determine ipilimumab’s ultimate role in the adjuvant setting for patients with melanoma.

Conclusion

The FDA approval of ipilimumab has re-energized the field of immunotherapy, and over 20,000 patients have been treated with CTLA-4–blocking antibodies to date. However, the full potential of ipilimumab and tremelimumab remains to be realized in the developing landscape of other novel immunotherapeutic approaches. It may ultimately be shown that CTLA-4 functions best as a component of combinatorial regimens. Further, as is covered in detail by Drs. Teply and Lipson in this special supplement to ONCOLOGY,[89] toxicity remains an impediment to the successful implementation of immune checkpoint blockade. In addition to prospective trials, preclinical models and biomarker research will be vital in the attempt to design rational combinations and to personalize therapy.

Acknowledgment:The authors wish to thank Dmitriy Zamarin for reviewing the figure and sidebar used in this article.

Financial Disclosure:Dr. Wolchok serves as a consultant to Bristol-Myers Squibb, Merck, MedImmune, and GlaxoSmithKline. Dr. Postow has received a research grant from Bristol-Myers Squibb, and is a consultant to and on the advisory board of Amgen. Drs. Funt and Page have no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article.

References:

1. Schwartz RH. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell. 1992;71:1065-8.

2. Krummel MF, Allison JP. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J Exp Med. 1995;182:459-65.

3. Linsley PS, Brady W, Urnes M, et al. CTLA-4 is a second receptor for the B cell activation antigen B7. J Exp Med. 1991;174:561-9.

4. Krummel MF, Allison JP. CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells. J Exp Med. 1996;183:2533-40.

5. Wing K, Onishi Y, Prieto-Martin P, et al. CTLA-4 control over Foxp3+ regulatory T cell function. Science. 2008;322:271-5.

6. Peggs KS, Quezada SA, Chambers CA, et al. Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti–CTLA-4 antibodies. J Exp Med. 2009;206:1717-25.

7. Simpson TR, Li F, Montalvo-Ortiz W, et al. Fc-
dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti–CTLA-4 therapy against melanoma. J Exp Med. 2013;210:1695-710.

8. Tivol EA, Borriello F, Schweitzer AN, et al. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity. 1995;3:541-7.

9. Waterhouse P, Penninger JM, Timms E, et al. Lymphoproliferative disorders with early lethality in mice deficient in CTLA-4. Science. 1995;270:985-8.

10. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science. 1996;271:1734-6.

11. Weber JS, Kähler KC, Hauschild A. Management of immune-related adverse events and kinetics of response with ipilimumab. J Clin Oncol. 2012;30:2691-7.

12. Wolchok JD, Hoos A, O’Day S, et al. Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria. Clin Cancer Res. 2009;15:7412-20.

13. 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.

14. Ribas A, Kefford R, Marshall MA, et al. Phase III randomized clinical trial comparing tremelimumab with standard-of-care chemotherapy in patients with advanced melanoma. J Clin Oncol. 2013;31:616-22.

15. Tarhini AA. Tremelimumab: a review of development to date in solid tumors. Immunotherapy. 2013;5:215-29.

16. Calabrò L, Morra A, Fonsatti E, et al. Tremelimumab for patients with chemotherapy-resistant advanced malignant mesothelioma: an open-label, single-arm, phase 2 trial. Lancet Oncol. 2013;14:1104-11.

17. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949-54.

18. Curtin JA, Fridlyand J, Kageshita T, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005;353:2135-47.

19. Shahabi V, Whitney G, Hamid O, et al. Assessment of association between BRAF-V600E mutation status in melanomas and clinical response to ipilimumab. Cancer Immunol Immunother. 2012;61:733-7.

20. Johnson DB, Lovly CM, Flavin M, et al. NRAS mutation: a potential biomarker of clinical response to immune-based therapies in metastatic melanoma (MM). J Clin Oncol. 2013;31(suppl):abstr 9019.

21. Dong Y, Richards J-A, Gupta R, et al. PTEN functions as a melanoma tumor suppressor by promoting host immune response. Oncogene. 2014;33:4632-42.

22. Alexandrov LB, Nik-Zainal S, Wedge DC, et al. Signatures of mutational processes in human cancer. Nature. 2013;500:415-21.

23. Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome landscapes. Science. 2013;339:1546-58.

24. 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-e442.

25. Charen AS, Makarov V, Merghoub T, et al. The neoantigen landscape underlying clinical response to ipilimumab. J Clin Oncol. 2014;32(suppl 5S):abstr 3003.

26. Adaniel C, Rendleman J, Polsky D, et al. Germline genetic determinants of immunotherapy response in metastatic melanoma. J Clin Oncol. 2014;32(suppl 5S): abstr 3004.

27. Cha E, Klinger M, Hou Y, et al. Improved survival with T cell clonotype stability after anti–CTLA-4 treatment in cancer patients. Sci Transl Med. 2014;6:238ra70.

28. Green DR, Ferguson T, Zitvogel L, et al. Immunogenic and tolerogenic cell death. Nat Rev Immunol. 2009;9:353-63.

29. Shurin MR. Dual role of immunomodulation by anticancer chemotherapy. Nat Med. 2013;19:20-2.

30. Casares N, Pequignot MO, Tesniere A, et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J Exp Med. 2005;202:1691-701.

31. Emens LA, Jaffee EM. Leveraging the activity of tumor vaccines with cytotoxic chemotherapy. Cancer Res. 2005;65:8059-64.

32. Tanaka F, Yamaguchi H, Ohta M, et al. Intratumoral injection of dendritic cells after treatment of anticancer drugs induces tumor-specific antitumor effect in vivo. Int J Cancer. 2002;101:265-9.

33. Vincent J, Mignot G, Chalmin F, et al. 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell–dependent antitumor immunity. Cancer Res. 2010;70:3052-61.

34. Suzuki E, Kapoor V, Jassar AS, et al. Gemcitabine selectively eliminates splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor-bearing animals and enhances antitumor immune activity. Clin Cancer Res. 2005;11:6713-21.

35. Kitano S, Postow MA, Ziegler CGK, et al. Computational algorithm-driven evaluation of monocytic myeloid-derived suppressor cell frequency for prediction of clinical outcomes. Cancer Immunol Res. 2014;8:812-21.

36. Weide B, Martens A, Zelba H, et al. Myeloid-derived suppressor cells predict survival of patients with advanced melanoma: comparison with regulatory T cells and NY-ESO-1- or melan-A–specific T cells. Clin Cancer Res. 2014;20:1601-9.

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

38. Weber J, Hamid O, Amin A, et al. Randomized phase I pharmacokinetic study of ipilimumab with or without one of two different chemotherapy regimens in patients with untreated advanced melanoma. Cancer Immun. 2013;13:7.

39. Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011; 364:2507-16.

40. 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.

41. 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.

42. Ruffini E, Asioli S, Filosso PL, et al. Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms. Ann Thorac Surg. 2009;87:365-71.

43. Kim KB, Sosman JA, Fruehauf JP, et al. BEAM: a randomized phase II study evaluating the activity of bevacizumab in combination with carboplatin plus paclitaxel in patients with previously untreated advanced melanoma. J Clin Oncol. 2012;30:34-41.

44. Oyama T, Ran S, Ishida T, et al. Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-kappa B activation in hemopoietic progenitor cells. J Immunol. 1998;160:1224-32.

45. Dikov MM, Ohm JE, Ray N, et al. Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation. J Immunol. 2005;174:215-22.

46. Yuan J, Zhou J, Dong Z, et al. Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab. Cancer Immunol Res. 2014;2:127-32.

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

48. 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.

49. Barker CA, Postow MA, Khan SA, et al. Concurrent radiotherapy and ipilimumab immunotherapy for patients with melanoma. Cancer Immunol Res. 2013;1:92-8.

50. Waitz R, Solomon SB, Petre EN, et al. Potent induction of tumor immunity by combining tumor cryoablation with anti–CTLA-4 therapy. Cancer Res. 2012;72:430-9.

51. Slovin SF, Higano CS, Hamid O, et al. Ipilimumab alone or in combination with radiotherapy in metastatic castration-resistant prostate cancer: results from an open-label, multicenter phase I/II study. Ann Oncol. 2013;24:1813-21.

52. Kwon ED, Drake CG, Scher HI, et al. Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after docetaxel chemotherapy (CA184-043): a multicentre, randomised, double-blind, phase 3 trial. Lancet Oncol. 2014;15:700-12.

53. Dhodapkar MV, Sznol M, Zhao B, et al. Induction of antigen-specific immunity with a vaccine targeting NY-ESO-1 to the dendritic cell receptor DEC-205. Sci Transl Med. 2014;6:232ra51.

54. Neyns B, Wilgenhof S, Corthals J, et al. Phase II study of autologous mRNA electroporated dendritic cells (TriMixDC-MEL) in combination with ipilimumab in patients with pretreated advanced melanoma.
J Clin Oncol. 2014;32(suppl 5S):abstr 3014.

55. Le DT, Lutz E, Uram JN, et al. Evaluation of ipilimumab in combination with allogeneic pancreatic tumor cells transfected with a GM-CSF gene in previously treated pancreatic cancer. J Immunother. 2013;36:382-9.

56. van Elsas A, Hurwitz AA, Allison JP. Combination immunotherapy of B16 melanoma using anti–cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med. 1999;190:355-66.

57. Hodi FS, Lee SJ, McDermott DF, et al. Multicenter, randomized phase II trial of GM-CSF (GM) plus ipilimumab (Ipi) versus Ipi alone in metastatic melanoma: E1608. J Clin Oncol. 2013;31(suppl):abstr CRA9007.

58. Hauschild A, Grob J-J, Demidov LV, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet. 2012;380(9839):358-65.

59. Flaherty KT, Infante JR, Daud A, et al. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med. 2012;367:1694-703.

60. Hu-Lieskovan S, Robert L, Moreno BH, et al. Combining targeted therapy with immunotherapy in BRAF-mutant melanoma: promise and challenges.
J Clin Oncol. 2014;32:2248-54.

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

62. Puzanov I, Callahan MK, Linette GP, et al. Phase 1 study of the BRAF inhibitor dabrafenib (D) with or without the MEK inhibitor trametinib (T) in combination with ipilimumab (Ipi) for V600E/K mutation–positive unresectable or metastatic melanoma (MM).
J Clin Oncol. 2014;32(suppl 5S):abstr 2511.

63. Ackerman A, Klein O, McDermott DF, et al. Outcomes of patients with metastatic melanoma treated with immunotherapy prior to or after BRAF inhibitors. Cancer. 2014;120:1695-701.

64. Frederick DT, Piris A, Cogdill AP, et al. BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma. Clin Cancer Res. 2013;19:1225-31.

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

66. Ribas A. Tumor immunotherapy directed at PD-1. N Engl J Med. 2012;366:2517-9.

67. Sznol M, Kluger HM, Callahan MK, et al. Survival, response duration, and activity by BRAF mutation (MT) status of nivolumab (NIVO, anti-PD-1, BMS-936558, ONO-4538) and ipilimumab (IPI) concurrent therapy in advanced melanoma (MEL). J Clin Oncol. 2014;32(suppl 5S):abstr LBA9003.

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

69. 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.

70. Melero I, Shuford WW, Newby SA, et al. Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat Med. 1997;3:682-5.

71. Sznol M, Hodi FS, Margolin K, et al. Phase I study of BMS-663513, a fully human anti-CD137 agonist monoclonal antibody, in patients (pts) with advanced cancer (CA). J Clin Oncol. 2008;26(15 suppl):abstr 3007.

72. Segal NH, Gopal AK, Bhatia S, et al. A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer. J Clin Oncol. 2014;32(suppl 5S):abstr 3007.

73. Kocak E, Lute K, Chang X, et al. Combination therapy with anti–CTL antigen-4 and anti-4-1BB antibodies enhances cancer immunity and reduces autoimmunity. Cancer Res. 2006;66:7276-84.

74. Belcaid Z, Phallen JA, Zeng J, et al. Focal radiation therapy combined with 4-1BB activation and CTLA-4 blockade yields long-term survival and a protective antigen-specific memory response in a murine glioma model. PLoS One. 2014;9:e101764.

75. Sugamura K, Ishii N, Weinberg AD. Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40. Nat Rev Immunol. 2004;4:420-31.

76. Curti BD, Kovacsovics-Bankowski M, Morris N, et al. OX40 is a potent immune-stimulating target in late-stage cancer patients. Cancer Res. 2013;73:7189-98.

77. Redmond WL, Linch SN, Kasiewicz MJ. Combined targeting of costimulatory (OX40) and coinhibitory (CTLA-4) pathways elicits potent effector t cells capable of driving robust antitumor immunity. Cancer Immunol Res. 2014;2:142-53.

78. Ribas A, Comin-Anduix B, Economou JS, et al. Intratumoral immune cell infiltrates, FoxP3, and indoleamine 2,3-dioxygenase in patients with melanoma undergoing CTLA4 blockade. Clin Cancer Res. 2009;15:390-9.

79. Hamid O, Schmidt H, Nissan A, et al. A prospective phase II trial exploring the association between tumor microenvironment biomarkers and clinical activity of ipilimumab in advanced melanoma. J Transl Med. 2011;9:204.

80. 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.

81. 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.

82. Hanks BA, Holtzhausen A, Evans KS, et al. Type III TGF-β receptor downregulation generates an immunotolerant tumor microenvironment. J Clin Invest. 2013;123:3925-40.

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

84. Hanks BA, Holtzhausen A, Evans K, et al. Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTEN-/- transgenic model of melanoma.
J Clin Oncol. 2014;32(suppl 5S):abstr 3011.

85. Parham P. MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol. 2005;5:201-14.

86. Wolchok JD, Neyns B, Linette G, et al. Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, double-blind, multicentre, phase 2, dose-ranging study. Lancet Oncol. 2010;11:155-64.

87. Antonia SJ, Gettinger SN, Chow LQM, et al. Nivolumab (anti-PD-1; BMS-936558, ONO-4538) and ipilimumab in first-line NSCLC: interim phase I results. J Clin Oncol. 2014;32(suppl 5S):abstr 8023.

88. Coit DG, Andtbacka R, Anker CJ, et al. Melanoma, version 2.2013: featured updates to the NCCN guidelines. J Natl Compr Canc Netw. 2013;11:395-407.

89. Teply BA, Lipson EJ. Identification and management of toxicities from immune checkpoint–blocking drugs. Oncology (Williston Park). 2014;28(suppl3)30-8.

90. Russell SJ, Peng K-W, Bell JC. Oncolytic virotherapy. Nat Biotechnol. 2012;30:658-70.

91. Schirrmacher V, Fournier P. Newcastle disease virus: a promising vector for viral therapy, immune therapy, and gene therapy of cancer. Methods Mol Biol. 2009;542:565-605.

92. Kaufman HL, Kim DW, DeRaffele G, et al. Local and distant immunity induced by intralesional vaccination with an oncolytic herpes virus encoding GM-CSF in patients with stage IIIc and IV melanoma. Ann Surg Oncol. 2010;17:718-30.

93. Zamarin D, Holmgaard RB, Subudhi SK, et al. Localized oncolytic virotherapy overcomes systemic tumor resistance to immune checkpoint blockade immunotherapy. Sci Transl Med. 2014;6:226ra32.

94. Puzanov I, Milhem MM, Andtbacka RHI, et al. Primary analysis of a phase 1b multicenter trial to evaluate safety and efficacy of talimogene laherparepvec (T-VEC) and ipilimumab (ipi) in previously untreated, unresected stage IIIB-IV melanoma. J Clin Oncol. 2014;32(suppl 5S):abstr 9029.