Clinical deployment of antibodies for treatment of melanoma

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Molecular Immunology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Molecular Immunology journal homepage: www.elsevier.com/locate/molimm

Review

Clinical deployment of antibodies for treatment of melanoma夽 Brendan D. Curti a , Walter J. Urba a,∗ a

Earle A. Chiles Research Institute, Providence Cancer Center, 4805 NE Glisan St. 2N35, Portland, OR 97213, United States

a r t i c l e

i n f o

Article history: Received 4 December 2014 Received in revised form 23 January 2015 Accepted 26 January 2015 Available online xxx Keywords: Melanoma CTLA-4 PD-1 OX40 4-1BB

a b s t r a c t The concept of using immunotherapy to treat melanoma has existed for decades. The rationale comes from the knowledge that many patients with melanoma have endogenous immune responses against their tumor cells and clinically meaningful tumor regression can be achieved in a minority of patients using cytokines such as interleukin-2 and adoptive cellular therapy. In the last 5 years there has been a revolution in the clinical management of melanoma in large measure based on the development of antibodies that influence T cell regulatory pathways by overcoming checkpoint inhibition and providing co-stimulation, either of which results in significantly more effective immune-mediated tumor destruction. This review will describe the pre-clinical and clinical application of antagonistic antibodies targeting the T-cell checkpoints cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), and agonistic antibodies targeting the costimulatory pathways OX40 and 4-1BB. Recent progress and opportunities for future investigation of combination antibody therapy will be described. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The humoral immune system is capable of making antibodies diverse enough to recognize over 10 billion foreign antigens with targets as diverse as microbial pathogens and tumor cells. After binding to antigen, antibody effector function is mediated by the following: complement fixation, Fc receptor binding leading to degranulation of neutrophils, engagement of other immune cells with cytotoxic function, antibody-dependent cellular cytotoxicity (ADCC) or prevention of binding of the antigen to adhesion or signaling molecules. These events in turn can promote a variety of regulatory functions that modulate the immune response including immunoglobulin class switching, cytokine release, B-cell memory and feedback regulation that influences immune enhancement or suppression. The adaptability and diversity of this system is carefully regulated, and B cells that produce antibodies that bind to self-antigen are eliminated. Autoimmune disease with significant clinical consequences can occur in patients where there is failure to eliminate or regulate self-reactive B cells. One could conjecture that allo-antibodies with the ability to promote immunologic memory or cytotoxic function in the setting of chronic infection or

夽 This article belongs to Special Issue on Therapeutic Antibodies. ∗ Corresponding author at: Earle A. Chiles Research Institute, Providence Cancer Center, 4805 NE Glisan St. 2N35, Portland, OR 97213, United States. Tel.: +1 503 215 6588; fax: +1 503 215 6841. E-mail addresses: [email protected] (B.D. Curti), [email protected] (W.J. Urba).

malignancy by engaging stimulatory T-cell receptors or inhibiting T-cell check-point proteins might confer some evolutionary benefit, but no naturally occurring antibody has been described with these properties. Therapeutic antibodies have been used in medical care and research for decades, but in the last 15 years they have become commonplace in oncologic management. The majority of these monoclonal antibodies are antagonistic, and were engineered to block a protein antigen of interest or to induce ADCC. The value of this approach has been translated numerous times in clinical medicine. Some examples from the last 15 years in medical oncology include rituximab (used in CD20+ B-cell malignancies) (Davis et al., 2000), trastuzumab (erb-b2-overexpressing breast cancer) (Slamon et al., 2001) and cetuximab (tumors that have mutated EGFR, such as colon cancer and head and neck cancer) (Baselga et al., 2005; Cunningham et al., 2004). The mechanism of tumor elimination with these therapeutic agents is complex and likely includes antibody-dependent cellular cytotoxicity (ADCC) as well as inhibition of growth pathways salient to tumor growth (Arnould et al., 2006; Ferris et al., 2010). Another area of therapeutic investigation has been the use of antibodies as immunomodulators. Anti-CD3, an antibody that binds the CD3 component of the T-cell receptor (TCR), administered at high doses causes immunosuppression and can be used to treat allograft rejection in patients who have received solid organ or bone marrow transplants (Bluestone et al., 1993). Anti-CD3 at low doses has immune stimulating effects and can trigger T-cell activation through the zeta chain of the TCR (Urba et al., 1992). Although

http://dx.doi.org/10.1016/j.molimm.2015.01.025 0161-5890/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Curti, B.D., Urba, W.J., Clinical deployment of antibodies for treatment of melanoma. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.01.025

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Fig. 1. Summary of major T-cell regulatory pathways and therapeutic targets. Antagonists of the T-cell checkpoints CTLA-4 and PD-1 or PD-L1 increase T cell activation and have antitumor activity in melanoma as well as other solid tumors. Agonists of the co-stimulatory OX40 and 4-1BB pathways also increase T-cell proliferation, effector function and memory.

anti-CD3 is not currently used as an anti-cancer agent, the observation that a T-cell-directed antibody could trigger T-cell cytotoxicity was an important conceptual insight that has lead to a proliferation of research on antibodies that influence the activation state and behavior of T cells resulting in enhanced anti-tumor immune responses. The CD3-zeta chain component has been used as a component of chimeric antigen receptors (CARs) to target tumor antigens with remarkable clinical activity in acute myelogenous leukemia and may have application in a wide variety of malignancies (Mardiros et al., 2013). Bispecific antibodies that link VH and VL for CD3 binding with the VH and VL that binds a tumor-associated antigen (e.g. CD19) have been used to redirect T cells to kill tumor cells. This approach has been studied in hematological malignancy and has recently garnered FDA approval, (Topp et al., 2014) but could also be applied to other solid tumors including melanoma. A rapidly expanding knowledge of the receptors and pathways that regulate T cells, natural killer (NK) cells and antigen-presenting cells (APC) has identified the targets to which the current generation of melanoma therapeutic antibodies has been engineered. The T-cell pathways that have been most extensively studied to develop therapeutic antibodies in cancer are the T-cell checkpoints known as cytotoxic T lymphocyte antigen-4 (CTLA-4 designated CD152) and programmed death-1 (PD1 designated CD279). In addition, the study of the costimulatory pathways OX40 (CD134) and 4-1BB (CD137) may also yield therapeutic advances (Fig. 1). This review will describe the T-cell pathways and therapeutic antibodies that are transforming the care of patients with melanoma and other cancers, the rationale for combination antibody therapies currently undergoing clinical investigation and future questions that need to be answered to optimize antibody-based cancer therapy. Although the pathways will be presented individually, it is important to recognize that they function concurrently and that the final effect on T-cell activation is the result of the integration of the multiple coinhibitory and costimulatory signals. 2. CTLA-4: Pre-clinical observations The steps that lead to T-cell activation include peptide antigen presentation by an antigen presenting cell (APC) to the TCR in the context of the appropriate major histocompatibility

complex (MHC) class molecule (signal 1) and engagement of a costimulatory receptor (signal 2), the most important of which is mediated through the interaction of CD28 on T cells and CD80/CD86 on APC. Other cytokine signals from APC or regulatory T cells can amplify or diminish immune responses (signal 3). T-cell activation also triggers pathways that eventually dampen the immune response. The chief regulatory pathway that shuts down a T-cell response after activation is CTLA-4, which is normally stored in vesicles in the cytosol of T cells and is released to the surface after antigen presentation (Alegre et al., 1996), where it out-competes CD28 for the binding of CD80/CD86. The net effect is to diminish signaling from the T-cell receptor to the nucleus. The observation that CTLA-4 mediates suppression of T cells led to the hypothesis that blocking its interaction with CD80 would increase T-cell activation and enhance anti-tumor immune responses, a hypothesis that was first investigated in the laboratory of Dr. James Allison (Leach et al., 1996). His group tested antagonistic antibodies to CTLA-4 in a variety of murine tumor models and demonstrated tumor regression and cures in some mice after anti-CTLA-4 administration. It is interesting to note that prolonged stability of some murine tumors and measurable but slow growth of others were described in this manuscript, an observation that is relevant given the clinical results that will be detailed below. These pre-clinical results illustrated a novel approach to cancer immunotherapy, which was robust and appeared simpler to administer in comparison with other immunotherapies under development at that time including adoptive cellular immunotherapy, vaccines and cytokines to promote an immune response. 3. Anti-CTLA4 clinical results Two anti-CTLA-4 antibodies, ipilimumab and tremelimumab, have undergone extensive clinical evaluation. Ipilimumab is a fully human IgG1 monoclonal antibody that binds to the CTLA-4 receptor expressed on activated T cells. Phase I and II studies of ipilimumab established a biologically active and tolerable dose; although a new class of immune-related toxicities were observed (see below). These early studies also established that patients with advanced melanoma had more objective tumor regressions, although tumor

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B.D. Curti, W.J. Urba / Molecular Immunology xxx (2015) xxx–xxx Table 1 Results of the pivotal trial leading to FDA approval of ipilimumab.

Survival at 1 year Survival at 2 years Complete and partial response (%) Disease control rate (%)* P value A versus C P value B versus C *

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Table 2 Summary of long-term results for ipilimumab in metastatic melanoma.

Ipilimumab + gp100

Ipilimumab + placebo

gp100 + placebo

44 22 5.7 20.1 0.0179 0.0002

46 24 10.9 28.5

25 14 1.5 11

Includes complete responses, partial responses and stable disease.

regression was also observed in patients with ovarian cancer, renal cancer, non-Hodgkin lymphoma and lung cancer (Ansell et al., 2009; Hodi et al., 2003; Yang et al., 2007). In addition, there was evidence of CD4+ and CD8+ T-cell recognition of melanocytes and extensive necrosis with immune infiltration of tumor sites in some in patients with melanoma. Two phase III randomized studies with ipilimumab were performed in patients with advanced melanoma (Robert et al., 2011; Hodi et al., 2010). The first study was conducted in patients with widely metastatic melanoma who expressed the HLA-A*0201 phenotype and had failed prior systemic therapies. The eligibility criterion for HLA-A*0201 expression was to allow comparison of ipilimumab to a control gp100 peptide vaccine. The specific gp100 peptides that comprised the vaccine are recognized only in the context of HLA-A*0201. Patients were assigned randomly to treatment groups in a proportion of 3:1:1 to ipilimumab (3 mg/kg IV q 3 weeks × 4 doses) and gp100 vaccine, ipilimumab monotherapy + placebo, or gp100 vaccine monotherapy + placebo, respectively. The objective response rates were low, but there was a statistically significant improvement in survival in the patients who received ipilimumab (Table 1). In a second phase III study, patients with advanced melanoma were randomized in a 1:1 proportion to ipilimumab (10 mg/kg IV q 3 weeks × 4 doses) and dacarbazine or dacarbazine and placebo as first line therapy (Robert et al., 2011). Overall survival was better in the patients receiving ipilimumab and dacarbazine (11.1 months versus 9.1 months for dacarbazine) and a higher proportion of patients in the ipilimumab group were alive at one, two and three years. Ipilimumab toxicities have been extensively reviewed (Fecher et al., 2013; Weber et al., 2012); side effects include rash, diarrhea, colitis with perforation, endocrinopathies, hepatocellular injury, fatigue and pyrexia. The side effects usually begin after the 2nd or 3rd dose and are presumed due to T-cell activation induced by CTLA-4 blockade by ipilimumab, although the precise mechanism is not well understood. Immune-mediated toxicity was not observed in preclinical murine or non-human primate models. Toxicities are ameliorated in the majority of patients by immunosuppressive agents including steroids, monoclonal antibodies that inhibit tumor necrosis factor (TNFi) and mycophenylate mofetil. Steroids are used first and often diminish toxicity within a week, after which the steroid can be tapered. The starting dose of corticosteroids for moderate immune-mediated toxicity is 0.5 mg/kg of prednisone (or equivalent). A dose of 1–2 mg/kg daily is recommended for severe immune-mediated toxicities. When the toxicity improves to a mild level, then a taper over 4 weeks should ensue. Ipilimumab treatment can often be continued after the steroid taper. Steroids do not seem to diminish the therapeutic effect of ipilimumab (Weber et al., 2009). TNFi therapy is most often employed to manage steroid-resistant immune-mediated diarrhea or colitis, while mycophenylate mofetil can be used to ameliorate immunemediated hepatic injury. There have been several longer term follow-up studies of patients with advanced melanoma who have received ipilimumab

Schadendorf et al. Patients treated Median survival (months) Survival at 1 year (%) Survival at 2 years (%) Immune related toxicity (%)

1861 11.4 44 28 14

Ascierto et al. 833 7.2 35 20 33

(Ascierto et al., 2014; Lebbe et al., 2014; Schadendorf et al., 2015). They have confirmed the improvements in survival described in the original phase III studies, which are clinically meaningful for patients with metastatic melanoma for whom the expected median survival previously was 6–9 months (Table 2). They also confirmed relatively low objective response rates (13%), but higher percentages of stable disease. There is a plateau on the survival curve presented by Schadendorf, with 18% of patients remaining alive from 5 to 10 years after receiving ipilimumab. Lebbe and colleagues also observed durable melanoma regression with greater than 20% of patients alive after 5 years. Unlike chemotherapy, where tumor regression is usually evident in a few weeks, the regression of melanoma following treatment with ipilimumab often takes many weeks, and sometimes months after the therapy is completed. Delayed responses to ipilimumab or rapid progression followed by marked regression (so-called “pseudoprogression”) have also been reported (Pennock et al., 2012; Weber et al., 2012). This is in concert with the original observations of tumor response in murine malignancy after anti-CTLA-4 (Leach et al., 1996). The recognition of marked differences in tumor response kinetics after anti-CTLA-4 compared to chemotherapy and other immunotherapies has changed clinical practice. The realization that stable and even progressing malignancy can be associated with improved survival means that patients may not need a new therapy immediately at the first sign of melanoma growth, although the percentage of patients who have tumor progression followed by regression is less than 10%. These observations have led to alternate measurement rules to assess clinical response, known as immune related response criteria (irRC) (Hoos et al., 2010). Since the validity of these criteria are still being evaluated in clinical trials they are not commonly used in clinical practice. Delayed response and pseudoprogression after ipilimumab are well-recognized; however, melanoma progression requiring another systemic therapy is still the most common clinical scenario. It would be ideal to have robust predictive biomarkers to determine the best clinical approach, but at present careful assessment of the patient’s functional status, a frank discussion of goals and options and a physician experienced in immunotherapy response remain the best approach to navigate these complex clinical situations. Tremelimumab, which is a fully human IgG2 monoclonal antibody, has also been tested in patients with melanoma, but has not been approved thus far by the FDA or other regulatory bodies for cancer therapy. Tremelimumab has a longer plasma half-life compared to ipilimumab (22 days compared with 15.4 days) and the IgG2 subtype has less affinity for binding to Fc␥ receptor (Fc␥R) on macrophages. Fc␥R binding and its influence on ADCC may be important in CTLA-4 blockade (see below). The results of a phase III randomized comparison of tremelimumab (15 mg/kg IV every 90 days) to dacarbazine or temozolomide chemotherapy in patients with metastatic melanoma have been reported (Ribas et al., 2013). There was no difference comparing tremelimumab and chemotherapy in objective response (10.7 versus 9.8%) or overall survival (12.4 versus 10.7 months); however, response duration was significantly longer with tremelimumab (35.8 versus 13.7 months). The mechanisms for the prolonged stability or delayed regression of melanoma in some patients are not understood, but may

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relate to the T-cell infiltration of tumors after anti-CTLA-4 (Huang et al., 2011). The mechanism is likely complex as tumors from both responding and non-responding patients have increased CD8+ tumor-infiltrating lymphocytes (TIL) after ipilimumab, implying that the mechanism involves not only cell infiltration, but also interactions between different immune cell types and their activity. The TCR repertoire is a potential marker to assess the antimelanoma activity of effector CD8+ T cells. There is a broadening of the TCR repertoire after anti-CTLA-4 (Robert et al., 2015, 2014b) in peripheral blood T cells. The complementarity-determining region 3 (CDR3) of the TCR V-beta region was sequenced in the peripheral blood mononuclear cells from patients with melanoma at baseline, 30 and 60 days after receiving tremelimumab. There was a 30% increase in the diversity of TCR sequences in 19 of the 21 patients that was not observed in normal controls. There were no differences in TCR diversity comparing patients with melanoma regression or progression although this observation strongly suggests that anti-CTLA-4 significantly influenced immune responses. This immunological diversity could be operative in the enhanced survival and increased intervals of melanoma stability associated with anti-CTLA-4 therapy. Another potential way to assess the activity of T cells after anti-CTLA-4 is to examine CTLA-4 on Treg. CTLA-4 is also present on intratumoral regulatory T cells and antiCTLA-4 decreases the activity of Treg in murine models (Selby et al., 2014). Along with the decrease in intratumoral Treg is a concomitant increase in effector T cells. This change in the balance of T-cell subtypes may account for enhanced anti-tumor effect. The specific mechanism of Treg depletion in the tumor likely involves elimination of Treg by macrophages in the tumor via ADCC resulting in a significantly increased ratio of effector T cells to Treg in the tumor. The immune activation from CTLA-4 antagonists may also depend on binding affinity. Anti-CTLA4 antibodies that have stronger binding to Fc␥R have significantly greater anti-tumor effects in MCA38, CT26 and B16 murine tumor models (Selby et al., 2014; Simpson et al., 2013). Interestingly, this change in effector to Treg ratio does not occur in tumor draining lymph nodes. Another implication of this observation is that anti-CTLA-4 antibodies with less Fc␥R binding affinity such as tremelimumab may have less clinical activity, although the objective response of tremelimumab compared to ipilimumab in metastatic melanoma is essentially equal. There are no known biomarkers before treatment or immunological measures after treatment that predict clinical response to anti-CTLA-4. There is a great need to develop predictive tests to help clinical decision making. Predictive and post-treatment biomarkers undergoing assessment include intratumoral myeloid-derived suppressor cells, amplification of pre-existing humoral immune response to the antigen NY-ESO-1, assessment of regulatory T cells and increases in peripheral blood inducible costimulator (ICOS)expressing CD4+ T cells (Kitano et al., 2014; Postow et al., 2001; Tarhini et al., 2014). A recent report suggested that the genetic landscape of an individual patient’s melanoma may be used to predict the clinical benefit from anti-CTLA-4 treatment (Snyder et al., 2014). Melanomas are known to have a high number of mutations compared to other malignancies. These mutations could serve as a source of neo-antigens to which an immune response could be promoted. Whole exome sequencing identified specific tumor neopeptides whose predicted ability to bind MHC Class I molecules and stimulate T cells were most likely to experience clinical benefits. This provocative finding needs to be confirmed but suggests a path toward the development of useful biomarkers for immunotherapeutic agents. The Food and Drug Administration (FDA) approved ipilimumab in March 2011 for patients with metastatic melanoma or unresectable disease. This was the first approval of a medication that demonstrated a survival benefit in randomized phase III studies for patients with advanced unresectable or metastatic melanoma.

There is ongoing clinical investigation of the use of ipilimumab in the adjuvant setting for patients with resected stage III melanoma. The preliminary clinical results of a phase III study investigating adjuvant ipilimumab versus placebo in patients with stage III melanoma were presented recently (Eggermont et al., 2014). Although the duration of follow-up was relatively short (2.7 years), there was a statistically significant difference in relapse-free survival of 26.1 versus 17.1 months comparing ipilimumab to placebo. Another phase III study (ECOG 1609) comparing two dose levels of ipilimumab with interferon in stage III melanoma has completed enrollment. The clinical results of this study have not yet been presented. There are also phase III studies in progress of ipilimumab in other malignancies such as metastatic prostate cancer. Many combinations are also being investigated including checkpoint and co-stimulatory antibodies (see below), oncolytic viruses and cytokines such as interleukin-2. 4. PD-1: Pre-clinical observations The main pathway that negatively regulates the behavior of T cells in the periphery (e.g. at sites of chronic infection or tumors) is the programmed death-1 (PD-1) pathway. PD-1 is a transmembrane protein receptor up-regulated on T cells after TCR engagement. The ligands for this receptor, (PD-L1 (B7-H1) and PDL2 (B7-DC)), found on APC, macrophages, T cells and B cells are up-regulated at the time of T-cell activation. PD-L1 is also expressed by many tumors including melanoma (Haile et al., 2011). PD-1 can also interact with CD80, one of the ligands for CTLA-4, therefore expression of PD-1 can potentially reinforce the inhibitory signal from the CTLA-4 pathway (also known as coinhibition). PD-1 and PD-L1 expression are associated with anergy and exhaustion of T cells, especially in the setting of chronic antigen exposure that occurs during unresolved infection or malignancy (Barber et al., 2006). When PD-1 and PD-L1 engage, there is dephosphorylation of SHP-2 on the intracellular portion of PD-1. The dephosphorylated SHP-2 inhibits proximal signaling kinases associated with the TCR, which in turn decreases cytokine production and cytotoxicity (Freeman, 2008). Engagement of PD-1 via PD-L1 or PD-L2 limits T cell activity at sites of chronic infection and may decrease autoimmunity during immune response to pathogens. Regulatory T cells (Treg) have high expression of PD-1 at tumor sites and may exert other inhibitory influences on anti-tumor responses (Francisco et al., 2009). Antigen-specific CD8+ T cells that infiltrate tumor also have high levels of PD-1 expression (Ahmadzadeh et al., 2009). The cytotoxic CD8+ T cells exhibited decreased cytokine production and PD-1+ Treg also expressed the proliferation marker Ki-67. Proliferation of Treg in the tumor would be expected to further diminish anti-tumor responses. Peripheral blood T cells in the same patients from whom the TIL were studied did not show increased PD-1 expression. It is also known that many tumor histologies including melanoma, ovarian cancer, colon cancer and non-small cell lung cancer express PD-L1 (Dong et al., 2002). Thus, immune modulation by the tumor within the tumor microenvironment may promote survival of the malignancy and decrease the ability of cytotoxic T cells to eradicate the tumor. Blockade of PD1 or PD-L1 using antagonistic antibodies has been shown to enhance tumor responses in many murine tumor models and may be more effective in combination with anti-CTLA-4 (Iwai et al., 2002; Curran et al., 2010). These findings provided a strong rationale for the clinical translation of PD-1/PD-L1 axis blockade in patients with melanoma and other malignancies. 5. Anti-PD-1 clinical results The first anti-PD-1 antibody tested in patients with melanoma was MDX-1106, a fully human IgG4, now referred to as nivolumab

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(Brahmer et al., 2010). This antibody blocks the interaction between PD-L1 and PD-1 and also the interaction between PD-1 and CD80 found on B cells and macrophages whose normal function is to provide a costimulatory signal when it engages CD28 on activated T cells. A total of 39 patients participated in the phase I study, which also included other cancer types (advanced prostate cancer, colon cancer, renal cancer and non-small cell lung (NSCLC)). The antibody was administered at doses from 0.3 to 10 mg/kg and was well tolerated with what appeared to be a lower incidence of immune-mediated toxicities compared to ipilimumab. Partial responses were observed in patients with melanoma and renal cancer, and one complete response was documented in colon cancer. Similar to preclinical studies, biopsy specimens from this clinical trial showed expression of PD-L1 in the tumor, which the authors suggested correlated with response. A larger phase I study investigated nivolumab at doses from 0.1 to 10 mg/kg and enrolled 239 patients with advanced melanoma, renal cancer and NSCLC (Topalian et al., 2012). Twenty-six of the 94 melanoma patients (28%) had objective tumor regression and the progression-free survival was 41 weeks. In addition, 6 patients had stability of melanoma for more than 24 weeks. PDL1 expression was assessed in tumor biopsies from 42 patients; objective responses were observed in 9 of 25 patients whose tumors expressed PD-L1 and 0 of 17 patients whose tumors were PD-L1 negative. Subsequent studies have shown that response to anti-PD-1 is higher in patients whose tumor expresses PDL1, but responses have also been described in patients whose tumors are PD-L1 negative. Some of these conflicting findings may be due to dynamic expression of PD-L1 and differences in immunohistochemical assay technique. Quantification of immune cell infiltrates (immunoprofiling) and specifically, PD-L1 expression on immune cells at the infiltrating border of the tumor may also be useful in determining prognosis and the likelihood of treatment response (Ascierto et al., 2013). Others have also observed that the adaptive expression of PD-L1 and the presence of T cells in the tumor microenvironment indicates an immune reactive environment and is the strongest correlate of clinical response to PD-1 blockade (Ribas and Tumeh, 2014; Taube et al., 2014). This association was strongest in melanoma, but also was found in patients with renal cancer and non-small cell lung carcinoma. Nivolumab was compared to dacarbazine in a randomized study in which 418 patients with advanced BRAF wild type melanoma participated who had received no prior systemic therapy (Robert et al., 2015, 2014b). The group that received nivolumab (n = 210) had statistically significantly better objective response (40% versus 13.9%), survival at 1 year (72.9% versus 42.1%) and progressionfree survival (5.1 versus 2.2 months) compared to patients who received dacarbazine. PD-L1 status was assessed by immunohistochemistry and positivity was defined as 5% or greater tumor cells expressing PD-L1. The objective response of nivolumab in PD-L1+ tumors was 52.7% and 33.1% in patients with PD-L1− tumors. Survival was better in patients treated with nivolumab, regardless of PD-L1 expression status. There is an ongoing phase III randomized open-label study comparing nivolumab to investigator’s choice chemotherapy (dacarbazine or the combination of paclitaxel and carboplatin) in patients with advanced melanoma after progression on antiCTLA-4 and/or BRAF-targeted therapy in patients whose melanoma expressed a BRAF mutation (Weber et al., 2014). The preliminary results of this trial were presented recently and the objective response of patients receiving nivolumab was 32% versus 11% in patients receiving chemotherapy. The median duration of response was not reached in the group that received nivolumab and was 3.6 months in the chemotherapy group. The clinical activity of nivolumab was higher than chemotherapy regardless of BRAF

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mutation status, the response to anti-CTLA-4 or PD-L1 expression on the melanoma before therapy. Nivolumab was approved by the FDA in December, 2014, to treat advanced melanoma progression after ipilimumab or BRAF inhibitor. Another anti-PD-1 antagonistic antibody targeted to compete with the interaction between PD-1 and PD-L1 and PD-L2, known as pembrolizumab, has been studied in melanoma (Robert et al., 2014a). The antibody is also a fully human IgG4. 173 patients with unresectable or metastatic melanoma who had disease progression after having received at least 2 doses of ipilimumab received pembrolizumab at 2 mg/kg (N = 89) or 10 mg/kg (N = 84). The objective response was 26% at both dose levels with a median time to response of 12 weeks. The median duration of response was not reached at the time of the publication. Immune-mediated toxicities were also observed, but with a lower severity and incidence compared to anti-CTLA-4. Fatigue (33%), pruritis and rash were the most common toxicities and did not differ in severity or incidence when the 2 and 10 mg/kg dose levels were compared. Although this study showed impressive clinical activity of pembrolizumab when used after ipilimumab, its use, and that of other PD-1-directed antibodies, in the first line in metastatic disease is undergoing further study. For instance, nivolumab used with or without a peptide vaccine against NY-ESO-1 showed anti-melanoma activity in patients who had not received ipilimumab and also those who had progression after ipilimumab (Weber et al., 2013). Other significant areas for clinical investigation include the optimal duration of treatment, further understanding of the time to change to a new therapy when there are mixed responses (e.g. regression of some tumor deposits and growth of others) and the potential application of anti-PD-1 in the adjuvant setting. The FDA approved pembrolizumab in September 2014 for the treatment of patients with advanced melanoma progressive after ipilimumab or BRAF-targeted therapy in patients whose melanomas express a BRAF mutation. There are now greater than 85 clinical trials studying anti-PD-1 or anti-PD-L1 monotherapy or combinations in patients with metastatic melanoma, bladder cancer, non-small cell lung cancer and renal cancer that may result in additional indications for this immunotherapy. An antibody targeting PD-L1 has also been tested in patients with melanoma and other advanced cancers (Brahmer et al., 2012). This IgG4 antibody was tested at doses between 0.3 and 10 mg/kg. Among the 207 patients who participated in the study, 55 had advanced melanoma. Objective response was observed in 9 of 52 melanoma patients who received 3 or more anti-PD-L1 doses (17%). An additional 14 patients (27%) had stable disease for at least 24 weeks. MPDL3280A is another anti-PD-L1 antibody that has entered clinical testing in melanoma and other malignancies (Hamid et al., 2013). This IgG1 antibody was engineered to decrease ADCC. The reason that this feature was incorporated into the antibody design was to address the concern that IgG4 antibodies that target the PD-1 pathway stimulate ADCC and could result in the elimination of activated T cells to which an IgG4 PD-1 pathway antagonist is bound. Clinical results of MPDL3280A in melanoma have only been presented in abstract form, but are encouraging. Toxicities were generally mild and no immune mediated colitis or pneumonitis was observed. The objective response in 41 patients with metastatic melanoma was 29%. Responses were observed in metastatic cutaneous and mucosal melanoma, but not in metastatic ocular melanoma. PD-L1 expression was assessed by immunohistochemistry (IHC). 27% of patients (4 out of 15) whose melanomas were PD-L1+ achieved an objective response while 20% of patients (3 out of 15) whose melanomas were PD-L1− responded. Specimens were not available in the other patients for PD-L1 assessment. A more comprehensive analysis of PD-L1 expression in the tumors of patients with a variety of cancer diagnoses who participated in

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Table 3 Duration of response in patients receiving concurrent ipilimumab and nivolumab. Nivolumab + Ipilimumab Regimen (mg/kg)

1-Year overall survival (%)

2-year overall survival (%)

Median overall survival (months)

Median progression-free survival (weeks)

85

79

40

27

57 94 94 100

50 – – –

27 NR NR NR

13 58 34 34

All concurrent (N = 53) 0.3 + 3 (14) 1 + 3 (17) 3 + 1 (16) 3 + 3 (6) NR = not reached, – = datum not reported.

MPDL3280A clinical trials has been published (Herbst et al., 2014). The expression of PD-L1 on TIL was a stronger correlate of response compared to PD-L1 expression on tumor. Other response correlates included the CTLA-4 expression on TIL, TH1 gene expression patterns and the absence of CX3CL-1 expression at baseline. These data support a growing consensus that the response to T-cell checkpoint antibodies depends on a pre-existing immune response against the tumor and that these agents help to overcome tumor-induced immune suppression.

There are many potential regulatory and co-stimulatory pathways that could enhance tumor response and many other potential combinations using vaccines, cytokines, radiation and therapies that target growth pathways that may be altered or up-regulated due to mutation. As of November 2014, there were 830 clinical trials listed on the National Cancer Institute website under the search term “combination immunotherapy” and 55 of these trials were for patients with melanoma.

6. Combination antibody therapy

7. Monoclonal antibodies under development for the treatment of melanoma and other solid tumors

As detailed above, monotherapy using antagonistic antibodies to CTLA-4, PD-1 or PD-L1 can induce significant tumor regressions and improve survival in patients with melanoma as well as other malignancies. There is also a strong pre-clinical rationale for blocking both CTLA-4 and PD-1 T-cell checkpoints in patients with melanoma. In a phase I study that investigated sequential and concurrent administration of ipilimumab and nivolumab (Wolchok et al., 2013). 17 of 53 patients received concurrent therapy at the maximum tolerated doses of ipilimumab (3 mg/kg) and nivolumab (1 mg/kg). The objective response in this group was 53%. Tumor regression occurred within the first 12 weeks in most of the responding patients; however, some individuals had initial growth of melanoma followed by subsequent shrinkage, similar to that described with ipilimumab monotherapy. The patients who participated in this study are still being followed for durability of response. The most recent response data from this study are summarized in Table 3. Treatment-related adverse events occurred in 93% of patients receiving combination immunotherapy. The most common side effects were rash (55%), pruritis (47%), fatigue (38%) and diarrhea (34%). Grade 3 or 4 adverse events related to treatment were observed in 49% of patients, but were generally reversible with steroids. The most common serious toxicities were hepatic (15%), gastrointestinal (9%), and renal (6%). These toxicities were deemed similar in severity and frequency to monotherapy with ipilimumab or nivolumab; however, a randomized phase III trial comparing ipilimumab, nivolumab and the combination that has recently completed accrual should define the objective response rate, toxicities and effects on overall survival of T-cell checkpoint monotherapy and combination therapy in melanoma.

Manipulation of other T-cell checkpoint and co-stimulatory pathways has high potential to improve melanoma therapy. Table 4 summarizes antibodies under development with application to melanoma. These pathways are of interest not only for their ability to promote T cell anti-tumor responses, but also because they are present in the TIL in melanoma and other tumors. Gros et al. (2014) reported on the extensive characterization of the TIL from 6 patients with melanoma. The expression of multiple T-cell checkpoint molecules was increased on melanoma-specific TIL including PD-1, lymphocyte-activated gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), and 4-1BB. The expression of PD-1 was most closely associated with clonal expansion of melanoma-specific T cells and this might be expected based on the normal function of PD-1 in immune regulation. Similar observations have been made in peripheral blood samples from patients with melanoma, wherein these investigators described the simultaneous co-expression of 4 or more inhibitory receptors on effector T cells, but only 1 or 2 inhibitory receptors were identified on naïve T cells (Baitsch et al., 2012). The recently developed oral agents that target BRAF mutations also alter immune cell infiltrates. Thirty-five melanoma specimens from 16 patients examined by immunohistochemistry (Frederick et al., 2013) showed an increase in the expression of PD-1, PD-L1 and TIM-3 on TIL in responding melanoma deposits. The extent to which these changes in TIL contribute to the melanoma responses induced by BRAF-targeted therapy is unknown. More discussion follows of the monoclonal antibodies for which there are published clinical data in patients with melanoma. The clinical development of these agents is important to the under-

Table 4 Therapeutic antibodies under clinical development in melanoma and other solid tumors. Main T-cell function or pathway Anti-OX40 (CD134) Anti-4-1BB (CD137) Anti-TIM-3 Anti-LAG-3 *

Survival, cytotoxicity and memory Survival and exhaustion Cytotoxicity and exhaustion Survival, cytoxicity and Treg function

Ab agonist or antagonist Agonist Agonist Antagonist Antagonist

Entered clinical testing as of November 2014 Yes Yes No Yes

Combinations under clinical investigation Anti-CTLA-4, anti-PD-L1, rituximab Anti-PD-1, anti-CS1, anti-KIR* – Anti-PD-1

KIR = killer inhibitory receptor.

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standing of immune system regulation and has many potential applications across solid tumor oncology. 8. OX40 OX40 is a member of the tumor necrosis factor receptor (TNFR) family that has a unique pattern of expression; it is for the most part restricted to lymphoid tissue and mainly expressed on activated CD4+ and CD8+ T cells (Baum et al., 1994; Paterson et al., 1987). Effector CD4+ T cells, which upregulate OX40 expression more rapidly than naïve T cells, express OX40 within 4 h after Ag stimulation (Gramaglia et al., 1998). OX40+ T cells are found preferentially at sites of inflammation and not normally in the peripheral blood. In animal models of autoimmunity and cancer, OX40+ T cells were enriched for the recently stimulated auto- or tumor Agreactive T cells (Buenafe et al., 1996; Weinberg et al., 1996, 2000). OX40-expressing TIL have been identified in human tumors and the tumor-draining lymph nodes in patients with melanoma and other solid tumors (Vetto et al., 1997). Injection of OX40 agonists leads to therapeutic responses in tumor-inoculated hosts in several preclinical murine cancer models including B16 melanoma (Weinberg et al., 2000). 9. Anti-OX40 in patients with advanced cancer A phase I trial tested three doses of a murine anti-human OX40 agonist antibody administered over five days (Curti et al., 2013). Thirty patients were treated with anti-OX40; ten at each dose level (0.1 mg/kg, 0.4 mg/kg and 2 mg/kg). Toxicities were mild; grade 1–2 fatigue and transient lymphopenia were the most common side effects observed. Toxicities generally resolved within 72 h of the last dose of anti-OX40. Immune-mediated diarrhea, colitis or endocrinopathies, which have been associated with ipilimumab, were not observed. A maximum tolerated dose was not reached within the dose range examined. The majority of patients had metastatic genitourinary malignancy (n = 10), melanoma (n = 7) or gastrointestinal cancer (n = 7). The best tumor response was stable disease using RECIST criteria. At least one tumor nodule regressed in 12 out of 30 patients including melanoma, renal cancer, prostate cancer, cholangiocarcinoma and squamous cell cancer of the urethra. Exploratory studies to look for tumor-specific immune responses were performed in four patients with melanoma. Peripheral blood mononuclear cells (PBMC) collected before and 57 days after anti-OX40 were co-cultured with autologous tumor, HLAmatched or HLA-mismatched melanoma cell lines for up to 5 days in four individuals for whom sufficient PBMC and/or autologous tumor were available. Between 2 and >10-fold increases in IFN-␥ levels were found in the supernatants after anti-OX40 in 3 out of 4 patients suggesting that a melanoma-specific immune response was induced by anti-OX40. The immune-enhancing properties of OX40 co-stimulation would be expected to amplify the anti-tumor effects of T-cell checkpoint inhibitors and several preclinical models have demonstrated this synergy. The combination of anti-OX40 (MEDI6469) and anti-CTLA-4 (tremelimumab) as well as anti-OX40 and anti-PDL1 (MEDI4736) are currently being investigated in a phase I clinical trial (NCT02205333). A human OX40 ligand:fusion protein agonist (MEDI6383) has also entered clinical trials. 10. 4-1BB Another important T cell co-stimulatory pathway is 4-1BB (CD137), also part of the TNFR family. When activated, 4-1BB induces Bcl2 and BCL-xl resulting in decreased T-cell apoptosis

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(Hernandez-Chacon et al., 2011). There is also enhanced tumorspecific T-cell cytotoxicity and NK activity after 4-1BB agonist administration. Anti-tumor activity has been observed in several murine models mediated by CD8+ T cells, involves interferongamma secretion and results in long-term memory against tumor antigen (Lin et al., 2010). 4-1BB is also expressed in tumor endothelium, DC, NK cells and hematopoietic cells. The expression of 4-1BB on endothelial cells may help to facilitate T-cell infiltration of tumor sites in conjunction with intracellular adhesion molecule-1 (ICAM1) and vascular adhesion molcule-1 (VCAM-1) (Palazon et al., 2011). A human agonistic anti-4-1BB (urelumab) has been tested in humans with advanced cancer as monotherapy and in combination with chemotherapy as well as anti-PD-1 (NCT02253992). Although rare, severe hepatic toxicity has been observed at urelumab doses greater than 1 mg/kg.

11. Conclusions Manipulation of the regulatory pathways that influence tumorreactive T cells has led to FDA approval of several new antibody monotherapies for patients with advanced melanoma. The investigation of inhibitory (checkpoint) and co-stimulatory pathways that influence T cell anti-tumor activity for clinical benefit is just beginning and appears to apply to many different cancers. The early results from combination antibody therapy attempting to influence both the CTLA-4 and PD-1 pathways are even more promising and are anticipated to change melanoma management and supersede monotherapy. The addition of co-stimulatory signals to T cells using agonists to OX40 or 4-1BB in conjunction with checkpoint inhibition could add significantly more anti-tumor efficacy based on pre-clinical models, but the degree to which these murine models will predict human tumor response is uncertain. There are many unanswered questions for the future of antibody therapy for use in melanoma. Although the FDA has approved pembrolizumab and nivolumab after ipilimumab failure, it is possible that the reverse sequence with blockade of PD-1 before CTLA-4 could yield better clinical results. The encouraging results of the combination of ipilimumab and nivolumab was a translation of murine models that showed synergy; however, there are many examples of pre-clinical anti-tumor activity using combinations that showed no benefit or unanticipated toxicity when tested in patients with cancer. The courage of many melanoma patients who volunteer for clinical trials has been invaluable in the development of T-cell antibody therapy, but most of these patients are not cured and they participate sequentially in clinical trials when the melanoma progresses. These prior treatments will likely influence the results of future therapies, but in ways that we cannot fully anticipate. This is a tremendous opportunity to learn more about the mechanisms of immunological resistance and the ways in which these antibodies alter the tumor microenvironment. It may be that any melanoma treatment acts as a selective pressure on the evolution of the melanoma, but there is no unified systematic analysis or panel of biomarkers to capture changes in immune response or the melanoma genome over time in individual patients. It is likely that there is great heterogeneity in the T-cell infiltrates of melanoma tumors within the same patient and across patients. A treatment sequence that works in one individual (or in an individual tumor) could be very different from another. Personalizing immunotherapy based on quantification of immune infiltrates (“immunoscore”) may help to optimize treatment choices and sequencing in individual patients (Ascierto et al., 2013). The antibody-based therapies for melanoma detailed in this review have occurred in a broader context of advances in the understanding of the major growth signaling pathways operative

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in melanoma. The development of inhibitors of mitogen-activated protein kinase (MAPK) components BRAF and MEK has also transformed the care of patients with advanced melanoma. These agents in combination have a high objective response and enhance survival; however, they do not cure melanoma (Long et al., 2014 and Larkin et al., 2014). There is a robust rationale for combining BRAF and MEK inhibitors with T-cell directed antibodies. There is an increase in CD8+ TIL that occurs early after beginning BRAF targeted therapy, (Wilmott et al., 2012) which could augment the effects of anti-CTLA-4 and anti-PD1. Radiation can also exert immunomodulatory effects and merits further study in combination with T-cell checkpoint antibodies and other immunotherapy in melanoma (Postow et al., 2012; Seung et al., 2012). 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Please cite this article in press as: Curti, B.D., Urba, W.J., Clinical deployment of antibodies for treatment of melanoma. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.01.025