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Emerging immunotherapy strategies in breast cancer Although immunogenicity is typically associated with renal cell carcinomas and melanoma, there are several compelling reasons why immune-based therapies should be explored in breast cancer. First, breast cancers express multiple putative tumor-associated antigens, such as HER‑2 and MUC‑1, which have been the successful focus of vaccine development over the past decade, translating into tumor-specific immune responses and, in some cases, clinical benefit. Second, passive immune strategies with anti-HER‑2 antibodies, such as trastuzumab and pertuzumab, have led to survival benefits in breast cancer. Finally, the successes observed with novel immunotherapeutic strategies, such as immune checkpoint blockade and adoptive T-cell therapies in other malignancies, combined with a growing body of literature that supports an interplay between solid tumors and the immune system, indicate that these strategies have the potential to revolutionize the treatment of breast cancer. KEYWORDS: adoptive therapy n breast cancer n immunotherapy n ipilimumab n nivolumab n trastuzumab n tremelimumab
After decades of investigation, the field of immunotherapy is experiencing a renaissance, rapidly transforming oncologic care across a variety of malignancies. For example, in 2011, a novel T-cell stimulating monoclonal antibody, called ipilimumab (Yervoy ®; Bristol-Myers Squibb, NY, USA), was granted US FDA approval for the treatment of metastatic malignant melanoma after demonstrating survival benefits in Phase III clinical trials [1,2]. Similarly, an autologous cellular vaccine, called sipuleucel-T (Provenge®; Dendreon, WA, USA), was FDA-approved after Phase III data demonstrated a survival benefit in prostate cancer [3]. The field of breast cancer is not naive to this renaissance. While not commonly cited as an immunotherapy, trastuzumab (Herceptin®; Genentech, CA, USA) – the most frequently used therapeutic antibody in breast oncology – functions as a potent stimulator of antibody-dependent cell-mediated cytotoxicity (ADCC) and T-cell-mediated tumor clearance, and has translated into significant clinical benefits for women with HER‑2-‘positive’ disease [4]. The relatively recent success of trastuzumab serves to foreshadow a possible revolution in the treatment of breast cancer. We anticipate that in the upcoming decade, previously developed and novel immunotherapeutic techniques may find new roles in the treatment of metastatic breast cancer (MBC), as well as in the prevention of recurrence in the adjuvant setting. In this review, we outline three distinct and promising
immunotherapeutic strategies in breast cancer and forecast future directions in the field.
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HER‑2-directed antibody: the first major inroad in breast cancer immunotherapy Anti-HER‑2 antibodies as immunotherapy HER‑2 is a proto-oncogene member of the EGF receptor family which serves as a therapeutic target for HER‑2-overexpressing (HER‑2+) breast cancers. Clinically available anti-HER‑2 antibody agents include trastuzumab, pertuzumab (Perjeta®; Genentech, CA, USA), and trastuzumab emtansine (T‑DM1, Kadcyla®; Genentech, CA, USA). The classical teaching is that anti-HER‑2 antibodies exert their clinical activity by blocking downstream growth signaling [4]. As a monoclonal antibody, it was anticipated that anti-HER‑2 antibodies could promote ADCC, a form of immune-mediated tumor death. In ADCC, the antigen-binding domain (Fab) of the antibody binds to the target cancer cell, whereas the constant domain (Fc) of the antibody binds to Fc receptors (FcgRIIIa) found on cytotoxic immune effector cells, such as NK cells. These receptor–antibody interactions mediate cytotoxic programs, including IFN‑g secretion and toxic granule release. In humans receiving anti-HER‑2 antibodies, this phenomenon has been well documented: trastuzumab administration increases levels of NK ADCC effector cells by flow cytometry, as well as induces
David B Page*1, Jarushka Naidoo1 & Heather L McArthur1 Memorial Sloan-Kettering Cancer Center, Department of Medicine, 300 East 66th Street, New York, NY 10065, NY, USA *Author for correspondence: Tel: +1 646 888 5440 [email protected] 1
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ADCC in 83% of subjects [5]. Some studies have identified a correlation between increases in NK cell activity and clinical outcome measures, such as progression-free survival [6,7]. Conversely, Fc receptor deficiencies and dysfunctional Fc polymorphisms have been associated with impaired response to trastuzumab [8,9]. Finally, the combination of trastuzumab with pertuzumab has been shown to generate synergistic NK cellmediated ADCC, capable of killing breast cancer stem cells (CD44hiCD24lowHER‑2low) that have been implicated in drug resistance and late relapse [10]. While the discovery of ADCC with trastuzumab was expected, it was not anticipated that the immune effects of anti-HER‑2 therapy would extend far beyond ADCC. More recent murine experiments have demonstrated that T-cell depletion impairs response to anti-HER‑2 therapy [11]. Similarly, blockade of key proteins of innate immune response signaling, including Myd88 and HMGB1, abrogates response to anti-HER‑2 therapy [11]. Finally, anti-HER‑2 therapy was shown to stimulate HER‑2 antibody production, as well as antibodies against unrelated antigens, such as IGFBP2 and p53. Thus, while classically construed as an inhibitor of growth signaling, trastuzumab and other anti-HER‑2 antibodies can now be considered bona fide immunotherapies, capable of orchestrating innate, adaptive and humoral immune responses [12,13]. Novel uses of existing anti-HER‑2 antibodies as ‘immunotherapies’ In light of these findings, clinicians have capitalized upon this identity of anti-HER‑2 antibody as ‘immunotherapy’. A natural next step was to combine anti-HER‑2 antibodies with other immunotherapeutic strategies. Indeed, preclinical data confirmed that concomitant administration of IL‑2 or IL‑12 with trastuzumab enhanced NK cell-mediated ADCC [14,15]. These strategies were subsequently evaluated in humans in several clinical trials. The first Phase I trial combined trastuzumab with IL‑2: the regimen was deemed safe and was capable of expanding NK cells and enhancing ADCC [16]. However, a follow-up Phase II study failed to demonstrate clinical response or ADCC/NK cell expansion in 13 tested subjects [17]. One possible explanation for this failure is that IL‑2 also induces expansion of suppressive Tregs [18]. A similar strategy of combining IL‑12 with trastuzumab was evaluated in 15 subjects: only three subjects experienced clinical benefit 196
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(one complete response and two stable disease); however, these subjects exhibited sustained NK cell-mediated IFN‑γ production [19]. A followup Phase I trial of trastuzumab plus IL‑12 plus paclitaxel appeared more promising, with five out of seven breast cancer subjects experiencing clinical benefit, and responders experiencing increased IFN‑g production [20]. In addition to evaluating novel combination regimens, the concept of coadministration with cytotoxic therapy has also been re-evaluated. The current standard of care in the setting of metastatic HER‑2+ breast cancer is to coadminister trastuzumab with cytotoxic therapy, based upon a randomized trial demonstrating survival benefit compared with chemotherapy [21]. However, recent data would suggest that cytotoxic therapy could impair the immunestimulating properties of anti-HER‑2 antibodies. When mice were treated with anti-HER‑2 antibody plus cyclophosphamide or paclitaxel, tumors diminished more rapidly compared with monotherapy. However, these mice remained susceptible to tumor rechallenge, whereas mice treated with anti-HER‑2 alone were capable of rejecting tumor rechallenges. This adverse effect occurred when chemotherapy was given 3 days after anti-HER‑2, whereas the ability to reject tumor was restored if chemotherapy was given the day prior to anti-HER‑2 therapy [11]. While preliminary, these studies suggest that alternative sequencing strategies may enhance the clinical activity of anti-HER‑2 therapy with cytotoxic chemotherapy. Developing the next generation of monoclonal antibody ‘immunotherapies’ Next-generation anti-HER‑2 antibodies have revolutionized the treatment of HER‑2+ breast cancer, as exemplified by the recent FDA approval of pertuzumab in the preoperative (neoadjuvant) setting [22] and trastuzumab–emtansine in the second-line metastatic setting [23]. Ongoing therapeutic innovations in HER‑2-targeted strategies are anticipated. However, one of the most significant drawbacks of antigen-directed passive immunotherapy is that only a minority of patients may express the target antigen; for example, with HER‑2 being overexpressed in only 20% of patients. Furthermore, tumors are heterogeneous, and prolonged therapy may select for overgrowth of antigen-negative clones. To that end, the development of novel antibodies for the treatment of HER‑2-normal disease is underway. Because a significant percentage of future science group
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HER‑2-normal tumors still express HER‑2 at low levels, an emerging strategy aims to target not the HER‑2 protein itself, but rather HER‑2 degradation products that are subsequently displayed on the cell surface, bound to the MHC. An antibody named RL1B has been developed that binds the HER‑2 E75 peptide–MHC class I complex with high affinity and specificity [24]. In a murine model, this antibody was capable of directly inducing cell apoptosis and lymphocytic tumor infiltrate, potentially mediated by ADCC and complement-dependent cytotoxicity [24]. These effects translated into a reduction in tumor growth and overall survival (OS) improvement in treated mice. This antibody serves as an example of a novel class of antibodies, called T-cell receptor mimics, that target oncogene peptides that are frequently expressed within MHC1, despite low-level surface expression of the oncogene itself. A second emerging antibody strategy abandons the HER‑2 target entirely and focuses on novel targets called human endogenous retrovirus elements (HERVs). HERVs are terminal repeat-like DNA sequences that constitute up to 8% of the human genome [25]. While transcriptionally silent in normal cells, the HERV‑K envelope protein is expressed in 66–86% of breast carcinomas versus 7% of normal breast tissue samples, and correlates with the presence of lymph node metastases [26,27]. Furthermore, spontaneous HERVK-specific antibody and T-cell responses are identifiable in HERV‑K-expressing breast cancer patients. In light of these data, an anti-HERV-K antibody was developed and evaluated for therapeutic potential. Treatment was shown to induce p53-mediated tumor apoptosis and reduce tumor size in both immunodeficient and immunocompetent xenograft mouse models [27]. This novel antibody strategy serves as just one example of how next-generation designer antibodies might shape the future treatment of breast cancer.
Playing detective: identifying & harnessing the next-generation vaccine target Vaccination is the longest-studied cancer immunotherapeutic strategy, researched since the 1910s when William Coley described the successful treatment of round-cell sarcoma with the intratumoral vaccination of Streptococcus and Serratia bacterial products [28]. While Coley’s vaccination utilized a nonspecific immune adjuvant, a successful vaccination strategy must both stimulate the immune system as well as direct it towards a viable tumor target. future science group
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It has been long known that patients may develop spontaneous immunity to HER‑2 in the form of antibody production and HER‑2-specific T-cell reactivity [29]. As such, the vast majority of early breast cancer vaccines have focused upon HER‑2, with varying success: HER‑2-directed vaccines can successfully augment a HER‑2-specific immune response and some HER‑2-directed vaccines have generated a signal of clinical benefit [30,31]. While HER‑2-directed vaccines may not be the panacea for every breast cancer patient, these efforts have facilitated our understanding of how to best design vaccines. Furthermore, significant progress has been made in discovering novel breast cancer vaccine targets. Here, we provide an overview of recent lessons learned with HER‑2 vaccines, as well as a summary of the emerging repertoire of novel breast tumor-associated antigens (TAAs), which may serve as targets for future vaccines. Lessons learned from HER‑2 vaccine development Numerous vaccination strategies against HER‑2 have been developed, including peptide-based, whole-protein, DNA-based, whole-tumor and dendritic cell (DC) vaccines. The most extensively studied vaccine is with GM‑CSF combined with E75, a HLA*A2/A3-restricted peptide derived from the HER‑2 extracellular domain. Recently, the 24‑month landmark analysis was published from the randomized Phase I/II US Military Cancer Institute Clinical Trials Group Studies I‑01 and I‑02, which evaluated the vaccine in 195 subjects with HER‑2-expressing tumors in the adjuvant setting [32]. In this trial, the 24‑month disease-free survival (DFS) was 90.2 versus 79.1% in the control group (p = 0.08), indicating a possibility of clinical efficacy that will be evaluated in a follow-up randomized trial. Another interesting feature of this trial was that late recurrences were associated with waning HER‑2-specific immunity. Thus, a booster vaccination program was initiated and subsequent subjects who received the booster demonstrated improved disease-free survival (p = 0.01). This study has taught us that booster injections may confer additive benefit. Recent investigations also suggest that concurrent cytotoxic therapy may enhance vaccine efficacy. While cytotoxic drugs have historically been considered immunosuppressive, emerging evidence demonstrates that cytotoxic therapy could have favorable immunomodulatory effects [33]. For example, lymphodepletion may suppress tolerance and promote subsequent www.futuremedicine.com
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antigen-specific T-cell responses [34]. Cytotoxic therapy may promote antigen presentation, NK-cell activation, innate immunity via Tolllike receptor‑4 expression and depletion of suppressive immune cell populations, such as Tregs or myeloid-derived suppressive cells (MDSCs) [34–36]. In light of these findings, a vaccine trial evaluated a HER‑2-expressing GM‑CSFsecreting allogeneic tumor vaccine alone or in sequence with low-dose cyclophosphamide and doxorubicin in a Phase I trial of 28 subjects with stable MBC [37]. Subjects receiving higher than 200mg/m2 cyclophosphamide experienced impairment in vaccine-induced delayed-type hypersensitivity. Conversely, lower doses were found to enhance HER‑2 autoantibody production. Using regression analyses, the most immunogenic combination was found to be 200mg/m 2 cyclophosphamide plus 35mg/m 2 doxorubicin. Based upon these data, future trials evaluating the combination of chemotherapy plus vaccination are anticipated.
More recently, a combination poxviral-based vaccine, which contains transgenes for MUC1, CEA and three T-cell costimulatory molecules, was evaluated in 12 heavily pretreated metastatic breast cancer patients. One subject experienced an ongoing complete response lasting >37 months and four subjects experienced stable disease (4–10 months). Some subjects experienced increases in CD4+ activity, as well as MUC‑1 reactivity by ELISPOT assay [43]. MUC‑1 continues to be heavily investigated. Another emerging strategy is to capitalize upon aberrant conformal elements identified exclusively in cancer-specific MUC‑1 glycoforms. Using novel covalent linking strategies, a novel MUC1-derived peptide vaccine demonstrated superior antitumor immunity in a preclinical model [44]. Finally, other mucin members, such as MUC‑3, have been identified as possible prognostic markers in breast cancer and may serve as unique targets for immunotherapy [39]. Cancer–testis antigens
An expanding repertoire of breast tumor antigens Going forward, we anticipate that next-generation vaccinations will incorporate some of these strategies; for example, the administration of boosters or combination with chemotherapy. However, we also expect a broader range of vaccines that capitalize upon recently identified novel breast cancer antigens. The mucin family of glycoproteins
Mucin glycoproteins, including MUC‑1, are glycoproteins expressed by a variety of epithelial cell types and malignancies, and drive tumorigenesis by promoting cell adhesion, blocking apoptotic pathways, and modulating intracellular growth signaling [38]. There are several reasons why MUC‑1 might serve as an ideal vaccination target. First, it is highly expressed in the majority (91%) of breast cancers, often with aberrant subcellular localization, which has been associated with inferior OS in a review of 1447 consecutive breast cancer cases [39]. Second, spontaneous anti-MUC‑1 IgM and IgG autoantibody production has been associated with favorable outcomes, such as increased time to metastasis [40,41]. Preliminary MUC‑1-directed vaccines have been shown to be safe and occasionally effective at generating antigen-specific T-cell and antibody responses [38]. One adjuvant Phase III trial of a mannan-conjugated MUC‑1 peptide vaccine demonstrated a 0% recurrence rate at 5.5 years compared with 27% in the placebo arm [42]. 198
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Cancer–testis (CT) antigens are predominantly expressed in human germline cells and have little or no expression in normal tissues, but nevertheless are expressed in a variety of malignancies and have been associated with high-grade lesions and poor outcomes [45]. Recent analyses in breast cancer revealed high expression of multiple CT antigens (7.3–26.7%) among ER- breast cancers, the most common being MAGEA and NY‑ESO‑1 [46]. Transfection of ER-expressing plasmids into NY‑ESO‑1+ER- cell lines does not appear to abrograte NY‑ESO‑1 expression, suggesting that CT antigen expression may be related to molecular subtype, rather than an inhibitory effect of ER [47]. Nevertheless, CT antigen expression does not appear to correlate with the basal-like molecular subtype, despite an association with high-grade ER- tumors [48,49]. The most investigated CT antigen is NY‑ESO‑1, expressed in 16% of triple-negative breast cancer and 2% of ER+/HER‑2- subjects. [50]. In NY‑ESO‑1-expressing breast cancer subjects, 73% were found to produce spontaneous NY‑ESO‑1 antibodies; these patients had higher CD8+ counts in the peripheral blood compared with negative patients [50], as well as more robust CD8+ T-cell and CD79a+ plasmocyte tumor infiltration [51]. Patients with NY‑ESO‑1 antibody production were more likely to have tumoral lymph node involvement, suggesting that tumoral infiltration may enhance the likelihood of an immune response [47]. In patients receiving ipilimumab, both NY‑ESO‑1 future science group
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antibody and T-cell reactivity were associated with an improved likelihood of clinical benefit [52]. Based upon these preclinical data, a Phase I evaluation of NY‑ESO‑1 vaccine ± sirolimus is accruing subjects with NY‑ESO‑1 expressing malignancies [201], as well as a Phase I study of NY‑ESO‑1 vaccine plus ipilimumab in melanoma [202]. NY‑BR‑1
Another putative breast cancer TAA is NY‑BR‑1, which was identified in 2001 and is detected in 60% of primary breast carcinomas, also expressed in normal mammary tissue [51]. In subjects with NY‑BR‑1-expressing tumors, 10% exhibit NY‑BR‑1-specific antibodies; NY‑BR‑1-specific T-cell recognition has also been identified. NY‑BR‑1 expression is correlated with ER positivity and estrogen response elements have been identified adjacent to the NY‑BR‑1 gene locus, suggesting that the protein is regulated by estrogen [53]. A retrospective evaluation of paired primary tumor/recurrences demonstrated that tamoxifen was associated with reduced NY‑BR‑1 expression [53]. NY‑BR‑1 serves as a potential vaccine target, but has yet to be evaluated in humans. P53
P53 serves as a favorable immunologic target in breast cancer, as mutations occur in up to 30% of breast cancers, prolonging the half-life of the protein and producing multiple immunogenic epitopes. Spontaneous p53-reactive T cells have been identified in more than 40% of breast cancer-treated patients; furthermore, the majority of breast cancer patients with high p53 expression are capable of mounting a p53-specific IFN‑g response [54]. Based on these data, a DC vaccine directed against p53 has been developed for clinical use. In a Phase II trial of a p53 DC vaccine in 26 subjects with metastatic disease, eight subjects attained stable disease and these subjects were more likely to mount a p53-specific T-cell response [55]. P53-directed vaccines are now being combined with other novel therapies, such as indoximod, an inhibitor of indolamine 2,3 dioxygenase. Indolamine 2,3 dioxygenase is overexpressed in tumors and blunts T-cell function by promoting enzymatic degradation of tryptophan in the tumor environment. In a Phase I study of a p53-directed dendritic cell vaccine in combination with indoximod, no objective responses were observed in 22 breast cancer patients; however, 60% of these patients responded to subsequent salvage future science group
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chemotherapy [56]. Therefore, a Phase II trial is planned in MBC [203].
From ‘Bacillus prodigiosus’ to ipilimumab: awakening the preconceived immune response A new class of immunotherapy: immune checkpoint therapy William Coley’s bacterial ‘Bacillus prodigiosus’ toxin is technically considered a vaccine because it was intended to induce an immune response against cancer. However, it differed from modern vaccines in that it lacked specificity for a tumor target, functioning instead to induce inflammation and potentiate pre-existing cancer immunity. Coley’s strategy now serves as a foundation for an emerging class of immunotherapies which function by augmenting preexisting immunity. Specifically, ‘immune checkpoint antibodies’ stimulate tumor recognition and clearance by binding key regulatory surface receptors on T cells and other immunologic cell populations, such as NK cells [57]. The first immune checkpoint target to be investigated was CTLA‑4, a coinhibitory T-cell receptor that is upregulated with T-cell activation and attenuates the immune response [58]. Ipilimumab and tremelimumab (Medimmune LLC, Gaithersburg, MD, USA) are human IgG monoclonal antibodies that bind CTLA‑4 and block downstream inhibitory signaling, and in doing so, reinvigorate an antitumor-specific T-cell response. Anti-CTLA‑4 antibodies were first investigated in malignant melanoma, where ipilimumab was found to improve OS, for the first time ever, in metastatic disease [1,2]. Anti-CTLA‑4 antibodies and other checkpoint antibodies are being actively investigated in breast cancer. A Phase I dose-escalation study of exemestane in combination with tremelimumab was deemed safe in 26 subjects, with only seven subjects experiencing grade III–IV treatment-related toxicities [59]. These included diarrhea/colitis (n = 4), lipase elevation (n = 1), dyspnea (n = 1) and rash (n = 1). One subject required hospitalization and treatment with infliximab for immune-mediated diarrhea, pyrexia and dehydration. The study determined the maximum tolerated dose for tremelimumab to be 6mg/kg intravenous every 90 days, and no dose-response correlation was identified. The best overall response was stable disease in 42% of patients (11/26), including patients who had received prior exemestane (four out of five). Durable response lasting a minimum of 12 weeks was achieved. The clinical benefit www.futuremedicine.com
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rate of exemestane monotherapy is 27% [60]. Immunologically, therapy was associated with increases in the ratio of effector T-cells:Tregs, as well as in the percentage of inducible costimulatory molecule-expressing T cells, a cell-surface marker associated with T-cell activation. The second published trial investigating immune checkpoint therapy in breast cancer is a Phase I/II study evaluating the combination of paclitaxel plus IMP321 [61]. IMP321 is a soluble form of LAG‑3, a protein expressed on activated T cells that binds MHC class II. In a preceding trial of IMP321 in metastatic renal cell carcinoma, IMP321 promoted favorable TH1 lineage differentiation, cytokine release, T-cell effector priming and lymph node migration [62]. In this trial, IMP321 was delivered subcutaneously every 2 weeks with weekly 80 mg/m 2 intravenous paclitaxel. This combination was safe in doses up to 6.25 mg, with no clinically significant IMP321-related adverse events reported. A 50% objective response was observed, compared with historical controls of 25% with single-agent paclitaxel. Additional evidence of incremental benefit was observed, including continued tumor regression during the maintenance phase after chemotherapy was completed. This effect was dose–dependent, reaching statistical significance at the 6.25‑mg dose. Immune monitoring revealed absolute increases in monocytes, DCs, NK cells, activated CD8+ cells and terminally differentiated effector memory cells (CD62L CD45RA+). Tumor regression was significantly correlated with increases in monocyte number and functionality. Novel checkpoint antibodies: the march toward cure in melanoma (& breast cancer?) Discovered in 1995, CTLA‑4 is certainly the prototypical immune checkpoint target [58]. However, since that time, multiple checkpoint molecules have been identified, resulting in explosive development of next-generation immune checkpoint antibodies [57]. One target of particular importance is PD‑1. This protein is expressed on a number of immune cell subsets, including activated T-cells, Tregs, activated B cells and NK cells. PD‑1 functions, in part, to regulate T cell activation in peripheral tissues [63]. Tumors may escape immune surveillance by expressing the endogenous ligand PD‑L1, which binds PD‑1 and suppresses antitumor activity of tumor-infiltrating lymphocytes. As such, blockade of the PD‑1 axis has been shown to enhance T-cell-mediated tumor clearance [63,64]. 200
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While early development of these next-generation checkpoint inhibitors was focused on malignant melanoma, we believe the dramatic success in melanoma may pave the way for subsequent development in breast carcinoma. The first anti-PD‑1 antibody to be tested in humans is nivolumab (Bristol Myers-Squibb, NY, USA). In the pivotal Phase I study, nivolumab produced a 31% objective response in melanoma, with a median OS of 16.8 months [65]. This compares favorably with a historical 6% objective response and 10.0‑month survival with ipilimumab [1,65]. In addition to the promise of heightened efficacy, the toxicity profile of nivolumab was favorable compared with ipilimumab; for example, with fewer subjects experiencing grade III/IV colitis/diarrhea. The next logical step was to evaluate the combination of ipilimumab + nivolumab in melanoma. In vitro combinations of ipilimumab plus nivolumab increased IFN‑g production sevenfold compared with monotherapy in a mixed lymphocyte reaction. In a murine model, combination therapy increased tumor-infiltrating effector lymphocytes and decreased infiltrating Tregs [66]. Based upon these data, ipilimumab plus nivolumab was evaluated in a Phase I trial in advanced melanoma [67]. At the maximum tolerated dose of ipilimumab 3 mg/kg and nivolumab 1 mg/kg, the response rate was a remarkable 53%, with all responding subjects experiencing a ≥80% decline in tumor burden at 12 weeks. The combination was safe, but with more frequent (53%) grade III/IV adverse events that were manageable with treatment algorithms. Thus, over the span of only 10 years, the drug class has radically transformed the treatment of melanoma from an invariably fatal disease to one with several active agents and the possibility of long-term durable remission. Preclinical data support the use of antiPD‑1/PD‑L1 antibodies in breast carcinoma. First, PD‑L1 is expressed in breast carcinomas [68]. PD‑L1-expression has been implicated as a possible biomarker for response to anti-PD‑1 therapy: in the nivolumab Phase I trial, patients with PD‑L1-expressing tumors had an objective response of 44 versus 17% among PD‑L1-negative patients [69]. However, in the ipilimumab/ nivolumab trial, responses were not dependent on PD‑L1-expression. Second, in a breast-tumor bearing hu‑SCID mouse model, antibody blockade of PD‑L1 plus DC vaccine prevented tumor growth and prolonged survival [70]. Finally, in transgenic mice models, combination therapy with anti-HER‑2 and -PD‑1 antibodies, as well future science group
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as combination of anti-HER‑2 and -CD137, were synergistic in promoting tumor regression [71,72]. These findings led to the development of a Phase I/II clinical trial evaluating nivolumab 1 mg/kg versus nivolumab 1 mg/kg ± ipilimumab 3 mg/kg in advanced triple-negative breast cancer patients [204]. To ensure tolerability of the combined regimen, a 3 + 3 dose-escalation runin will be performed first in the combination arm. In addition, we are participating in a trial of the anti-PD‑L1 inhibitor MEDI4736, evaluating monotherapy in advanced triple-negative breast cancer patients [205]. These studies will provide pivotal information on clinical efficacy and immune correlates in breast cancer, which will help to shape future studies. In the future, combinations such as anti-PD‑1 or -CD137 antibodies with anti-HER‑2 therapies, such as trastuzumab or trastuzumab emtansine, may be seen. While these therapies have been generally tolerable with preservation of quality of life while on therapy [73], they have also been associated with rare but life-threatening toxicities. For example, ipilimumab is associated with
Emerging immunotherapy strategies in breast cancer Although immunogenicity is typically associated with renal cell carcinomas and melanoma, there are several compelling reasons why immune-based therapies should be explored in breast cancer. First, breast cancers express multiple putative tumor-associated antigens, such as HER‑2 and MUC‑1, which have been the successful focus of vaccine development over the past decade, translating into tumor-specific immune responses and, in some cases, clinical benefit. Second, passive immune strategies with anti-HER‑2 antibodies, such as trastuzumab and pertuzumab, have led to survival benefits in breast cancer. Finally, the successes observed with novel immunotherapeutic strategies, such as immune checkpoint blockade and adoptive T-cell therapies in other malignancies, combined with a growing body of literature that supports an interplay between solid tumors and the immune system, indicate that these strategies have the potential to revolutionize the treatment of breast cancer. KEYWORDS: adoptive therapy n breast cancer n immunotherapy n ipilimumab n nivolumab n trastuzumab n tremelimumab
After decades of investigation, the field of immunotherapy is experiencing a renaissance, rapidly transforming oncologic care across a variety of malignancies. For example, in 2011, a novel T-cell stimulating monoclonal antibody, called ipilimumab (Yervoy ®; Bristol-Myers Squibb, NY, USA), was granted US FDA approval for the treatment of metastatic malignant melanoma after demonstrating survival benefits in Phase III clinical trials [1,2]. Similarly, an autologous cellular vaccine, called sipuleucel-T (Provenge®; Dendreon, WA, USA), was FDA-approved after Phase III data demonstrated a survival benefit in prostate cancer [3]. The field of breast cancer is not naive to this renaissance. While not commonly cited as an immunotherapy, trastuzumab (Herceptin®; Genentech, CA, USA) – the most frequently used therapeutic antibody in breast oncology – functions as a potent stimulator of antibody-dependent cell-mediated cytotoxicity (ADCC) and T-cell-mediated tumor clearance, and has translated into significant clinical benefits for women with HER‑2-‘positive’ disease [4]. The relatively recent success of trastuzumab serves to foreshadow a possible revolution in the treatment of breast cancer. We anticipate that in the upcoming decade, previously developed and novel immunotherapeutic techniques may find new roles in the treatment of metastatic breast cancer (MBC), as well as in the prevention of recurrence in the adjuvant setting. In this review, we outline three distinct and promising
immunotherapeutic strategies in breast cancer and forecast future directions in the field.
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Immunotherapy (2014) 6(2), 195–209
HER‑2-directed antibody: the first major inroad in breast cancer immunotherapy Anti-HER‑2 antibodies as immunotherapy HER‑2 is a proto-oncogene member of the EGF receptor family which serves as a therapeutic target for HER‑2-overexpressing (HER‑2+) breast cancers. Clinically available anti-HER‑2 antibody agents include trastuzumab, pertuzumab (Perjeta®; Genentech, CA, USA), and trastuzumab emtansine (T‑DM1, Kadcyla®; Genentech, CA, USA). The classical teaching is that anti-HER‑2 antibodies exert their clinical activity by blocking downstream growth signaling [4]. As a monoclonal antibody, it was anticipated that anti-HER‑2 antibodies could promote ADCC, a form of immune-mediated tumor death. In ADCC, the antigen-binding domain (Fab) of the antibody binds to the target cancer cell, whereas the constant domain (Fc) of the antibody binds to Fc receptors (FcgRIIIa) found on cytotoxic immune effector cells, such as NK cells. These receptor–antibody interactions mediate cytotoxic programs, including IFN‑g secretion and toxic granule release. In humans receiving anti-HER‑2 antibodies, this phenomenon has been well documented: trastuzumab administration increases levels of NK ADCC effector cells by flow cytometry, as well as induces
David B Page*1, Jarushka Naidoo1 & Heather L McArthur1 Memorial Sloan-Kettering Cancer Center, Department of Medicine, 300 East 66th Street, New York, NY 10065, NY, USA *Author for correspondence: Tel: +1 646 888 5440 [email protected] 1
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ADCC in 83% of subjects [5]. Some studies have identified a correlation between increases in NK cell activity and clinical outcome measures, such as progression-free survival [6,7]. Conversely, Fc receptor deficiencies and dysfunctional Fc polymorphisms have been associated with impaired response to trastuzumab [8,9]. Finally, the combination of trastuzumab with pertuzumab has been shown to generate synergistic NK cellmediated ADCC, capable of killing breast cancer stem cells (CD44hiCD24lowHER‑2low) that have been implicated in drug resistance and late relapse [10]. While the discovery of ADCC with trastuzumab was expected, it was not anticipated that the immune effects of anti-HER‑2 therapy would extend far beyond ADCC. More recent murine experiments have demonstrated that T-cell depletion impairs response to anti-HER‑2 therapy [11]. Similarly, blockade of key proteins of innate immune response signaling, including Myd88 and HMGB1, abrogates response to anti-HER‑2 therapy [11]. Finally, anti-HER‑2 therapy was shown to stimulate HER‑2 antibody production, as well as antibodies against unrelated antigens, such as IGFBP2 and p53. Thus, while classically construed as an inhibitor of growth signaling, trastuzumab and other anti-HER‑2 antibodies can now be considered bona fide immunotherapies, capable of orchestrating innate, adaptive and humoral immune responses [12,13]. Novel uses of existing anti-HER‑2 antibodies as ‘immunotherapies’ In light of these findings, clinicians have capitalized upon this identity of anti-HER‑2 antibody as ‘immunotherapy’. A natural next step was to combine anti-HER‑2 antibodies with other immunotherapeutic strategies. Indeed, preclinical data confirmed that concomitant administration of IL‑2 or IL‑12 with trastuzumab enhanced NK cell-mediated ADCC [14,15]. These strategies were subsequently evaluated in humans in several clinical trials. The first Phase I trial combined trastuzumab with IL‑2: the regimen was deemed safe and was capable of expanding NK cells and enhancing ADCC [16]. However, a follow-up Phase II study failed to demonstrate clinical response or ADCC/NK cell expansion in 13 tested subjects [17]. One possible explanation for this failure is that IL‑2 also induces expansion of suppressive Tregs [18]. A similar strategy of combining IL‑12 with trastuzumab was evaluated in 15 subjects: only three subjects experienced clinical benefit 196
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(one complete response and two stable disease); however, these subjects exhibited sustained NK cell-mediated IFN‑γ production [19]. A followup Phase I trial of trastuzumab plus IL‑12 plus paclitaxel appeared more promising, with five out of seven breast cancer subjects experiencing clinical benefit, and responders experiencing increased IFN‑g production [20]. In addition to evaluating novel combination regimens, the concept of coadministration with cytotoxic therapy has also been re-evaluated. The current standard of care in the setting of metastatic HER‑2+ breast cancer is to coadminister trastuzumab with cytotoxic therapy, based upon a randomized trial demonstrating survival benefit compared with chemotherapy [21]. However, recent data would suggest that cytotoxic therapy could impair the immunestimulating properties of anti-HER‑2 antibodies. When mice were treated with anti-HER‑2 antibody plus cyclophosphamide or paclitaxel, tumors diminished more rapidly compared with monotherapy. However, these mice remained susceptible to tumor rechallenge, whereas mice treated with anti-HER‑2 alone were capable of rejecting tumor rechallenges. This adverse effect occurred when chemotherapy was given 3 days after anti-HER‑2, whereas the ability to reject tumor was restored if chemotherapy was given the day prior to anti-HER‑2 therapy [11]. While preliminary, these studies suggest that alternative sequencing strategies may enhance the clinical activity of anti-HER‑2 therapy with cytotoxic chemotherapy. Developing the next generation of monoclonal antibody ‘immunotherapies’ Next-generation anti-HER‑2 antibodies have revolutionized the treatment of HER‑2+ breast cancer, as exemplified by the recent FDA approval of pertuzumab in the preoperative (neoadjuvant) setting [22] and trastuzumab–emtansine in the second-line metastatic setting [23]. Ongoing therapeutic innovations in HER‑2-targeted strategies are anticipated. However, one of the most significant drawbacks of antigen-directed passive immunotherapy is that only a minority of patients may express the target antigen; for example, with HER‑2 being overexpressed in only 20% of patients. Furthermore, tumors are heterogeneous, and prolonged therapy may select for overgrowth of antigen-negative clones. To that end, the development of novel antibodies for the treatment of HER‑2-normal disease is underway. Because a significant percentage of future science group
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HER‑2-normal tumors still express HER‑2 at low levels, an emerging strategy aims to target not the HER‑2 protein itself, but rather HER‑2 degradation products that are subsequently displayed on the cell surface, bound to the MHC. An antibody named RL1B has been developed that binds the HER‑2 E75 peptide–MHC class I complex with high affinity and specificity [24]. In a murine model, this antibody was capable of directly inducing cell apoptosis and lymphocytic tumor infiltrate, potentially mediated by ADCC and complement-dependent cytotoxicity [24]. These effects translated into a reduction in tumor growth and overall survival (OS) improvement in treated mice. This antibody serves as an example of a novel class of antibodies, called T-cell receptor mimics, that target oncogene peptides that are frequently expressed within MHC1, despite low-level surface expression of the oncogene itself. A second emerging antibody strategy abandons the HER‑2 target entirely and focuses on novel targets called human endogenous retrovirus elements (HERVs). HERVs are terminal repeat-like DNA sequences that constitute up to 8% of the human genome [25]. While transcriptionally silent in normal cells, the HERV‑K envelope protein is expressed in 66–86% of breast carcinomas versus 7% of normal breast tissue samples, and correlates with the presence of lymph node metastases [26,27]. Furthermore, spontaneous HERVK-specific antibody and T-cell responses are identifiable in HERV‑K-expressing breast cancer patients. In light of these data, an anti-HERV-K antibody was developed and evaluated for therapeutic potential. Treatment was shown to induce p53-mediated tumor apoptosis and reduce tumor size in both immunodeficient and immunocompetent xenograft mouse models [27]. This novel antibody strategy serves as just one example of how next-generation designer antibodies might shape the future treatment of breast cancer.
Playing detective: identifying & harnessing the next-generation vaccine target Vaccination is the longest-studied cancer immunotherapeutic strategy, researched since the 1910s when William Coley described the successful treatment of round-cell sarcoma with the intratumoral vaccination of Streptococcus and Serratia bacterial products [28]. While Coley’s vaccination utilized a nonspecific immune adjuvant, a successful vaccination strategy must both stimulate the immune system as well as direct it towards a viable tumor target. future science group
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It has been long known that patients may develop spontaneous immunity to HER‑2 in the form of antibody production and HER‑2-specific T-cell reactivity [29]. As such, the vast majority of early breast cancer vaccines have focused upon HER‑2, with varying success: HER‑2-directed vaccines can successfully augment a HER‑2-specific immune response and some HER‑2-directed vaccines have generated a signal of clinical benefit [30,31]. While HER‑2-directed vaccines may not be the panacea for every breast cancer patient, these efforts have facilitated our understanding of how to best design vaccines. Furthermore, significant progress has been made in discovering novel breast cancer vaccine targets. Here, we provide an overview of recent lessons learned with HER‑2 vaccines, as well as a summary of the emerging repertoire of novel breast tumor-associated antigens (TAAs), which may serve as targets for future vaccines. Lessons learned from HER‑2 vaccine development Numerous vaccination strategies against HER‑2 have been developed, including peptide-based, whole-protein, DNA-based, whole-tumor and dendritic cell (DC) vaccines. The most extensively studied vaccine is with GM‑CSF combined with E75, a HLA*A2/A3-restricted peptide derived from the HER‑2 extracellular domain. Recently, the 24‑month landmark analysis was published from the randomized Phase I/II US Military Cancer Institute Clinical Trials Group Studies I‑01 and I‑02, which evaluated the vaccine in 195 subjects with HER‑2-expressing tumors in the adjuvant setting [32]. In this trial, the 24‑month disease-free survival (DFS) was 90.2 versus 79.1% in the control group (p = 0.08), indicating a possibility of clinical efficacy that will be evaluated in a follow-up randomized trial. Another interesting feature of this trial was that late recurrences were associated with waning HER‑2-specific immunity. Thus, a booster vaccination program was initiated and subsequent subjects who received the booster demonstrated improved disease-free survival (p = 0.01). This study has taught us that booster injections may confer additive benefit. Recent investigations also suggest that concurrent cytotoxic therapy may enhance vaccine efficacy. While cytotoxic drugs have historically been considered immunosuppressive, emerging evidence demonstrates that cytotoxic therapy could have favorable immunomodulatory effects [33]. For example, lymphodepletion may suppress tolerance and promote subsequent www.futuremedicine.com
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antigen-specific T-cell responses [34]. Cytotoxic therapy may promote antigen presentation, NK-cell activation, innate immunity via Tolllike receptor‑4 expression and depletion of suppressive immune cell populations, such as Tregs or myeloid-derived suppressive cells (MDSCs) [34–36]. In light of these findings, a vaccine trial evaluated a HER‑2-expressing GM‑CSFsecreting allogeneic tumor vaccine alone or in sequence with low-dose cyclophosphamide and doxorubicin in a Phase I trial of 28 subjects with stable MBC [37]. Subjects receiving higher than 200mg/m2 cyclophosphamide experienced impairment in vaccine-induced delayed-type hypersensitivity. Conversely, lower doses were found to enhance HER‑2 autoantibody production. Using regression analyses, the most immunogenic combination was found to be 200mg/m 2 cyclophosphamide plus 35mg/m 2 doxorubicin. Based upon these data, future trials evaluating the combination of chemotherapy plus vaccination are anticipated.
More recently, a combination poxviral-based vaccine, which contains transgenes for MUC1, CEA and three T-cell costimulatory molecules, was evaluated in 12 heavily pretreated metastatic breast cancer patients. One subject experienced an ongoing complete response lasting >37 months and four subjects experienced stable disease (4–10 months). Some subjects experienced increases in CD4+ activity, as well as MUC‑1 reactivity by ELISPOT assay [43]. MUC‑1 continues to be heavily investigated. Another emerging strategy is to capitalize upon aberrant conformal elements identified exclusively in cancer-specific MUC‑1 glycoforms. Using novel covalent linking strategies, a novel MUC1-derived peptide vaccine demonstrated superior antitumor immunity in a preclinical model [44]. Finally, other mucin members, such as MUC‑3, have been identified as possible prognostic markers in breast cancer and may serve as unique targets for immunotherapy [39]. Cancer–testis antigens
An expanding repertoire of breast tumor antigens Going forward, we anticipate that next-generation vaccinations will incorporate some of these strategies; for example, the administration of boosters or combination with chemotherapy. However, we also expect a broader range of vaccines that capitalize upon recently identified novel breast cancer antigens. The mucin family of glycoproteins
Mucin glycoproteins, including MUC‑1, are glycoproteins expressed by a variety of epithelial cell types and malignancies, and drive tumorigenesis by promoting cell adhesion, blocking apoptotic pathways, and modulating intracellular growth signaling [38]. There are several reasons why MUC‑1 might serve as an ideal vaccination target. First, it is highly expressed in the majority (91%) of breast cancers, often with aberrant subcellular localization, which has been associated with inferior OS in a review of 1447 consecutive breast cancer cases [39]. Second, spontaneous anti-MUC‑1 IgM and IgG autoantibody production has been associated with favorable outcomes, such as increased time to metastasis [40,41]. Preliminary MUC‑1-directed vaccines have been shown to be safe and occasionally effective at generating antigen-specific T-cell and antibody responses [38]. One adjuvant Phase III trial of a mannan-conjugated MUC‑1 peptide vaccine demonstrated a 0% recurrence rate at 5.5 years compared with 27% in the placebo arm [42]. 198
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Cancer–testis (CT) antigens are predominantly expressed in human germline cells and have little or no expression in normal tissues, but nevertheless are expressed in a variety of malignancies and have been associated with high-grade lesions and poor outcomes [45]. Recent analyses in breast cancer revealed high expression of multiple CT antigens (7.3–26.7%) among ER- breast cancers, the most common being MAGEA and NY‑ESO‑1 [46]. Transfection of ER-expressing plasmids into NY‑ESO‑1+ER- cell lines does not appear to abrograte NY‑ESO‑1 expression, suggesting that CT antigen expression may be related to molecular subtype, rather than an inhibitory effect of ER [47]. Nevertheless, CT antigen expression does not appear to correlate with the basal-like molecular subtype, despite an association with high-grade ER- tumors [48,49]. The most investigated CT antigen is NY‑ESO‑1, expressed in 16% of triple-negative breast cancer and 2% of ER+/HER‑2- subjects. [50]. In NY‑ESO‑1-expressing breast cancer subjects, 73% were found to produce spontaneous NY‑ESO‑1 antibodies; these patients had higher CD8+ counts in the peripheral blood compared with negative patients [50], as well as more robust CD8+ T-cell and CD79a+ plasmocyte tumor infiltration [51]. Patients with NY‑ESO‑1 antibody production were more likely to have tumoral lymph node involvement, suggesting that tumoral infiltration may enhance the likelihood of an immune response [47]. In patients receiving ipilimumab, both NY‑ESO‑1 future science group
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antibody and T-cell reactivity were associated with an improved likelihood of clinical benefit [52]. Based upon these preclinical data, a Phase I evaluation of NY‑ESO‑1 vaccine ± sirolimus is accruing subjects with NY‑ESO‑1 expressing malignancies [201], as well as a Phase I study of NY‑ESO‑1 vaccine plus ipilimumab in melanoma [202]. NY‑BR‑1
Another putative breast cancer TAA is NY‑BR‑1, which was identified in 2001 and is detected in 60% of primary breast carcinomas, also expressed in normal mammary tissue [51]. In subjects with NY‑BR‑1-expressing tumors, 10% exhibit NY‑BR‑1-specific antibodies; NY‑BR‑1-specific T-cell recognition has also been identified. NY‑BR‑1 expression is correlated with ER positivity and estrogen response elements have been identified adjacent to the NY‑BR‑1 gene locus, suggesting that the protein is regulated by estrogen [53]. A retrospective evaluation of paired primary tumor/recurrences demonstrated that tamoxifen was associated with reduced NY‑BR‑1 expression [53]. NY‑BR‑1 serves as a potential vaccine target, but has yet to be evaluated in humans. P53
P53 serves as a favorable immunologic target in breast cancer, as mutations occur in up to 30% of breast cancers, prolonging the half-life of the protein and producing multiple immunogenic epitopes. Spontaneous p53-reactive T cells have been identified in more than 40% of breast cancer-treated patients; furthermore, the majority of breast cancer patients with high p53 expression are capable of mounting a p53-specific IFN‑g response [54]. Based on these data, a DC vaccine directed against p53 has been developed for clinical use. In a Phase II trial of a p53 DC vaccine in 26 subjects with metastatic disease, eight subjects attained stable disease and these subjects were more likely to mount a p53-specific T-cell response [55]. P53-directed vaccines are now being combined with other novel therapies, such as indoximod, an inhibitor of indolamine 2,3 dioxygenase. Indolamine 2,3 dioxygenase is overexpressed in tumors and blunts T-cell function by promoting enzymatic degradation of tryptophan in the tumor environment. In a Phase I study of a p53-directed dendritic cell vaccine in combination with indoximod, no objective responses were observed in 22 breast cancer patients; however, 60% of these patients responded to subsequent salvage future science group
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chemotherapy [56]. Therefore, a Phase II trial is planned in MBC [203].
From ‘Bacillus prodigiosus’ to ipilimumab: awakening the preconceived immune response A new class of immunotherapy: immune checkpoint therapy William Coley’s bacterial ‘Bacillus prodigiosus’ toxin is technically considered a vaccine because it was intended to induce an immune response against cancer. However, it differed from modern vaccines in that it lacked specificity for a tumor target, functioning instead to induce inflammation and potentiate pre-existing cancer immunity. Coley’s strategy now serves as a foundation for an emerging class of immunotherapies which function by augmenting preexisting immunity. Specifically, ‘immune checkpoint antibodies’ stimulate tumor recognition and clearance by binding key regulatory surface receptors on T cells and other immunologic cell populations, such as NK cells [57]. The first immune checkpoint target to be investigated was CTLA‑4, a coinhibitory T-cell receptor that is upregulated with T-cell activation and attenuates the immune response [58]. Ipilimumab and tremelimumab (Medimmune LLC, Gaithersburg, MD, USA) are human IgG monoclonal antibodies that bind CTLA‑4 and block downstream inhibitory signaling, and in doing so, reinvigorate an antitumor-specific T-cell response. Anti-CTLA‑4 antibodies were first investigated in malignant melanoma, where ipilimumab was found to improve OS, for the first time ever, in metastatic disease [1,2]. Anti-CTLA‑4 antibodies and other checkpoint antibodies are being actively investigated in breast cancer. A Phase I dose-escalation study of exemestane in combination with tremelimumab was deemed safe in 26 subjects, with only seven subjects experiencing grade III–IV treatment-related toxicities [59]. These included diarrhea/colitis (n = 4), lipase elevation (n = 1), dyspnea (n = 1) and rash (n = 1). One subject required hospitalization and treatment with infliximab for immune-mediated diarrhea, pyrexia and dehydration. The study determined the maximum tolerated dose for tremelimumab to be 6mg/kg intravenous every 90 days, and no dose-response correlation was identified. The best overall response was stable disease in 42% of patients (11/26), including patients who had received prior exemestane (four out of five). Durable response lasting a minimum of 12 weeks was achieved. The clinical benefit www.futuremedicine.com
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rate of exemestane monotherapy is 27% [60]. Immunologically, therapy was associated with increases in the ratio of effector T-cells:Tregs, as well as in the percentage of inducible costimulatory molecule-expressing T cells, a cell-surface marker associated with T-cell activation. The second published trial investigating immune checkpoint therapy in breast cancer is a Phase I/II study evaluating the combination of paclitaxel plus IMP321 [61]. IMP321 is a soluble form of LAG‑3, a protein expressed on activated T cells that binds MHC class II. In a preceding trial of IMP321 in metastatic renal cell carcinoma, IMP321 promoted favorable TH1 lineage differentiation, cytokine release, T-cell effector priming and lymph node migration [62]. In this trial, IMP321 was delivered subcutaneously every 2 weeks with weekly 80 mg/m 2 intravenous paclitaxel. This combination was safe in doses up to 6.25 mg, with no clinically significant IMP321-related adverse events reported. A 50% objective response was observed, compared with historical controls of 25% with single-agent paclitaxel. Additional evidence of incremental benefit was observed, including continued tumor regression during the maintenance phase after chemotherapy was completed. This effect was dose–dependent, reaching statistical significance at the 6.25‑mg dose. Immune monitoring revealed absolute increases in monocytes, DCs, NK cells, activated CD8+ cells and terminally differentiated effector memory cells (CD62L CD45RA+). Tumor regression was significantly correlated with increases in monocyte number and functionality. Novel checkpoint antibodies: the march toward cure in melanoma (& breast cancer?) Discovered in 1995, CTLA‑4 is certainly the prototypical immune checkpoint target [58]. However, since that time, multiple checkpoint molecules have been identified, resulting in explosive development of next-generation immune checkpoint antibodies [57]. One target of particular importance is PD‑1. This protein is expressed on a number of immune cell subsets, including activated T-cells, Tregs, activated B cells and NK cells. PD‑1 functions, in part, to regulate T cell activation in peripheral tissues [63]. Tumors may escape immune surveillance by expressing the endogenous ligand PD‑L1, which binds PD‑1 and suppresses antitumor activity of tumor-infiltrating lymphocytes. As such, blockade of the PD‑1 axis has been shown to enhance T-cell-mediated tumor clearance [63,64]. 200
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While early development of these next-generation checkpoint inhibitors was focused on malignant melanoma, we believe the dramatic success in melanoma may pave the way for subsequent development in breast carcinoma. The first anti-PD‑1 antibody to be tested in humans is nivolumab (Bristol Myers-Squibb, NY, USA). In the pivotal Phase I study, nivolumab produced a 31% objective response in melanoma, with a median OS of 16.8 months [65]. This compares favorably with a historical 6% objective response and 10.0‑month survival with ipilimumab [1,65]. In addition to the promise of heightened efficacy, the toxicity profile of nivolumab was favorable compared with ipilimumab; for example, with fewer subjects experiencing grade III/IV colitis/diarrhea. The next logical step was to evaluate the combination of ipilimumab + nivolumab in melanoma. In vitro combinations of ipilimumab plus nivolumab increased IFN‑g production sevenfold compared with monotherapy in a mixed lymphocyte reaction. In a murine model, combination therapy increased tumor-infiltrating effector lymphocytes and decreased infiltrating Tregs [66]. Based upon these data, ipilimumab plus nivolumab was evaluated in a Phase I trial in advanced melanoma [67]. At the maximum tolerated dose of ipilimumab 3 mg/kg and nivolumab 1 mg/kg, the response rate was a remarkable 53%, with all responding subjects experiencing a ≥80% decline in tumor burden at 12 weeks. The combination was safe, but with more frequent (53%) grade III/IV adverse events that were manageable with treatment algorithms. Thus, over the span of only 10 years, the drug class has radically transformed the treatment of melanoma from an invariably fatal disease to one with several active agents and the possibility of long-term durable remission. Preclinical data support the use of antiPD‑1/PD‑L1 antibodies in breast carcinoma. First, PD‑L1 is expressed in breast carcinomas [68]. PD‑L1-expression has been implicated as a possible biomarker for response to anti-PD‑1 therapy: in the nivolumab Phase I trial, patients with PD‑L1-expressing tumors had an objective response of 44 versus 17% among PD‑L1-negative patients [69]. However, in the ipilimumab/ nivolumab trial, responses were not dependent on PD‑L1-expression. Second, in a breast-tumor bearing hu‑SCID mouse model, antibody blockade of PD‑L1 plus DC vaccine prevented tumor growth and prolonged survival [70]. Finally, in transgenic mice models, combination therapy with anti-HER‑2 and -PD‑1 antibodies, as well future science group
Emerging immunotherapy strategies in breast cancer
as combination of anti-HER‑2 and -CD137, were synergistic in promoting tumor regression [71,72]. These findings led to the development of a Phase I/II clinical trial evaluating nivolumab 1 mg/kg versus nivolumab 1 mg/kg ± ipilimumab 3 mg/kg in advanced triple-negative breast cancer patients [204]. To ensure tolerability of the combined regimen, a 3 + 3 dose-escalation runin will be performed first in the combination arm. In addition, we are participating in a trial of the anti-PD‑L1 inhibitor MEDI4736, evaluating monotherapy in advanced triple-negative breast cancer patients [205]. These studies will provide pivotal information on clinical efficacy and immune correlates in breast cancer, which will help to shape future studies. In the future, combinations such as anti-PD‑1 or -CD137 antibodies with anti-HER‑2 therapies, such as trastuzumab or trastuzumab emtansine, may be seen. While these therapies have been generally tolerable with preservation of quality of life while on therapy [73], they have also been associated with rare but life-threatening toxicities. For example, ipilimumab is associated with
