Novel cancer antigens for personalized immunotherapies: latest evidence and clinical potential

Abstract

The clinical success of monoclonal antibody immune checkpoint modulators such as ipilimumab, which targets cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and the recently approved agents nivolumab and pembrolizumab, which target programmed cell death receptor 1 (PD-1), has stimulated renewed enthusiasm for anticancer immunotherapy, which was heralded by Science as ‘Breakthrough of the Year’ in 2013. As the potential of cancer immunotherapy has been recognized since the 1890s when William Coley showed that bacterial products could be beneficial in cancer patients, leveraging the immune system in the treatment of cancer is certainly not a new concept; however, earlier attempts to develop effective therapeutic vaccines and antibodies against solid tumors, for example, melanoma, frequently met with failure due in part to self-tolerance and the development of an immunosuppressive tumor microenvironment. Increased knowledge of the mechanisms through which cancer evades the immune system and the identification of tumor-associated antigens (TAAs) and negative immune checkpoint regulators have led to the development of vaccines and monoclonal antibodies targeting specific tumor antigens and immune checkpoints such as CTLA-4 and PD-1. This review first discusses the established targets of currently approved cancer immunotherapies and then focuses on investigational cancer antigens and their clinical potential. Because of the highly heterogeneous nature of tumors, effective anticancer immunotherapy-based treatment regimens will likely require a personalized combination of therapeutic vaccines, antibodies and chemotherapy that fit the specific biology of a patient’s disease.

Introduction

The possibility that immunotherapy could be useful for the treatment of cancer was first realized in the 1890s when William B. Coley showed that injecting killed bacterial cultures, known as Coley toxins, had beneficial effects in cancer patients [Nauts et al. 1953]. Decades later, studies performed in chemically induced sarcoma mouse models showed that syngeneic mice injected with irradiated sarcoma cells displayed immunity when later challenged with live sarcoma cells [Foley, 1953; Prehn and Main, 1957; Klein et al. 1960]. The fact that the immunity conferred in this manner was tumor-specific suggested that tumors express unique antigens that are specifically recognized by the adaptive immune system. Indeed, the first such tumor-associated antigen (TAA), known as melanoma antigen 1 (MAGE-1, also known as MAGE-A1), was identified in human melanoma cells by Boon and colleagues in 1991 [van der Bruggen et al. 1991]. Since that seminal discovery, the number of new TAAs has grown steadily to the point where there are now over 400 T-cell-defined human tumor antigenic peptides that have been identified [Vigneron et al. 2013]. Until the late 20th century, the role of immunosurveillance in cancer control had been the subject of much debate [Schreiber et al. 2011]; however, two studies by Schreiber and colleagues involving interferon gamma and tumor immunogenicity in immunocompetent hosts are considered to have played a major role in renewing interest in tumor immunology [Kaplan et al. 1998; Shankaran et al. 2001]. Over the years, a number of different approaches to cancer immunotherapy, including antibodies, cytokines, adoptive cell therapy (ACT) and therapeutic vaccines, have been attempted, but the overall response rates have been largely disappointing [Kirkwood et al. 2012; Savage et al. 2014]. Only recently, as knowledge of tumor biology and immunology has improved, has the complex nature of the interactions between the immune system and cancer come into focus, which has allowed the development of more specifically targeted agents [Kirkwood et al. 2012; Galluzzi et al. 2014].

Nonmutated, shared self-antigens constitute the majority of currently identified TAAs and can be classified into three major categories: (a) tumor-specific or cancer-testis antigens (CTAs), for example, MAGE-1, normally found only in the testes but are aberrantly expressed by a number of different cancers; (b) differentiation antigens expressed by both tumors and the normal differentiated cells from which the tumors arise, for example, melanoma antigen recognized by T cells (MART-1, also known as Melan-A); and (c) self-antigens that are overexpressed by tumors, for example, mucin 1 (MUC1) [Savage et al. 2014]. CTAs were the first type of TAA identified, and there are currently over 200 genes that have been classified as CTAs [Almeida et al. 2009]. It is believed that the observed antigenicity of CTAs is ascribed to the privileged immune status of the testis, where the blood–testis barrier prevents the entry of immune cells. Developing spermatozoa also do not express major histocompatibility complex (MHC) class I molecules, allowing them to evade immunosurveillance by infiltrating T cells [Whitehurst, 2014]. Thus, CTAs make attractive targets for immunotherapy; however, medullary thymic epithelial cells (mTECs) have been reported to express CTAs such as MAGE and New York esophageal squamous cell carcinoma 1 (NY-ESO-1) [Gotter et al. 2004], which suggests that central tolerance to CTAs can develop. A transcriptional regulator known as Aire (autoimmune regulator), which is expressed by mTECs, promotes the promiscuous expression of tissue-restricted antigens such as CTAs [Anderson et al. 2002; Derbinski et al. 2005], resulting in promotion of tolerance to TAAs through thymic deletion of self-reactive T cells [Savage et al. 2014].

In general, active immunotherapy of solid tumors requires the induction of cellular [T helper type 1 (T_H1) and cytotoxic T lymphocyte (CTL)-mediated] as opposed to humoral [T helper type 2 (T_H2), antibody-mediated] immune responses in order to be effective [Rosenberg, 2001; Kirkwood et al. 2012; Melero et al. 2014]. A T_H1-polarized immune response involving CTLs and natural killer (NK) cells mediates the elimination of tumor cells, while a T_H2-polarized immune response can have deleterious effects by promoting tumor development and progression [Kirkwood et al. 2012; Curigliano et al. 2013]. This is not to say that antibody-based anticancer immunotherapies are ineffective against solid tumors. Indeed, the past decade has seen the clinical development of numerous monoclonal antibodies directed at growth factors such as vascular endothelial growth factor A (VEGFA; e.g. bevacizumab), growth factor receptors such as human epidermal growth factor receptor (EGFR or HER1; e.g. cetuximab, panitumumab) and EGFR 2 (HER2; e.g. trastuzumab, pertuzumab), and negative immune checkpoint regulators such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4; e.g. ipilimumab) and programmed cell death receptor 1 (PD-1; e.g. nivolumab, pembrolizumab) [Sliwkowski and Mellman, 2013]. In addition to the direct, modulatory effects on signal transduction, antibody-dependent cellular cytotoxicity (ADCC), mediated through engagement of NK cells and macrophages, also likely contributes to the activity of these antibodies [Kohrt et al. 2012; Sliwkowski and Mellman, 2013]. The tumor-targeting monoclonal antibodies, perhaps the most commonly utilized form of anticancer immunotherapy [Weiner et al. 2010; Vacchelli et al. 2014], are examples of passive immunotherapy, while immune checkpoint inhibitors are considered to be active immunotherapies [Galluzzi et al. 2014].

The recent clinical development and approval of the immune checkpoint inhibitors ipilimumab, which targets CTLA-4, and nivolumab and pembrolizumab, both of which target PD-1, has generated a great deal of excitement in the field of cancer immunotherapy [Swaika et al. 2015]. In a phase III clinical trial, ipilimumab was the first immunotherapeutic to significantly improve overall survival (OS) in patients with advanced, metastatic melanoma [Hodi et al. 2010], and since then both nivolumab [Robert et al. 2015a] and pembrolizumab [Robert et al. 2015b] have demonstrated significant improvement in OS in this patient population. This review will first discuss CTLA-4, PD-1, and other established targets of cancer immunotherapy and then focus on novel tumor antigens and their clinical potential.

Established targets of anticancer immunotherapies

Currently approved anticancer immunotherapies, including monoclonal antibodies and cancer vaccines, target a number of different antigens including CTLA-4, PD-1, EGFR, HER2, VEGF, VEGF-R2, and PAP (prostatic acid phosphatase). Another well-known TAA is BCR-ABL (breakpoint cluster region Abelson tyrosine kinase), for which several small molecule tyrosine kinase inhibitors (TKIs) have been approved and for which immunotherapies are currently under development. Monoclonal antibodies are by far the most commonly approved form of antigen-specific anticancer immunotherapy, with only a single therapeutic cancer vaccine, sipuleucel-T (trade name Provenge^®), which targets the prostate cancer antigen PAP, having received Food and Drug Administration (FDA) approval to date. These immunotherapeutic targets and the clinical efficacy of approved treatments are discussed below. For a comprehensive list of the antigen targets of established anticancer immunotherapies, refer to Table 1.

Table 1.

Antigenic targets of approved immunotherapies.

Antigen	Cancer Types	Targeted Agents	References
BCR-ABL	CML ALL	Imatinib, Dasatinib Nilotinib, Bosutinib Ponatinib	[Isfort et al. 2014; Lindauer and Hochhaus, 2014; Ostendorf et al. 2014; Waller, 2014; Wehrle et al. 2014]
CD19	ALL	Blinatumomab	[Hoffman and Gore, 2014]
CD20	NHL, CLL B-cell NHL pre-B ALL	Rituximab Ofatumumab 90Y-Ibritumomab 131I-Tositumomab	[Sliwkowski and Mellman, 2013; Ai and Advani, 2015]
CD30	Hodgkin’s lymphoma	Brentuximab vedotin	[Sliwkowski and Mellman, 2013; Chen and Chen, 2015]
CD33	AML	Gemtuzumab ozogamicin	[Sliwkowski and Mellman, 2013; Loke et al. 2015]
CD52	CLL	Alemtuzumab	[Skoetz et al. 2012; Sliwkowski and Mellman, 2013]
CTLA-4	Unresectable or metastatic melanoma	Ipilimumab	[Hodi et al. 2010; Robert et al. 2011; Hodi et al. 2014]
EGFR	CRC Head and Neck	Cetuximab Panitumumab	[Cunningham et al. 2004; Saltz et al. 2004; Bonner et al. 2006; Van Cutsem et al. 2007; Vermorken et al. 2008; Van Cutsem et al. 2009; Douillard et al. 2010; Price et al. 2014]
EpCAM	Malignant ascites	Catumaxomab	[Seimetz, 2011]
HER2	Breast	Trastuzumab Pertuzumab	[Slamon et al. 2001; Romond et al. 2005; Bang et al. 2010; Perez et al. 2011; O’Sullivan and Connolly, 2014; Perez et al. 2014]
PAP	Prostate	Sipuleucel-T	[Kantoff et al. 2010a]
PD-1	Metastatic melanoma NSCLC	Nivolumab Pembrolizumab	[Topalian et al. 2012; Robert et al. 2014; Topalian et al. 2014; Garon et al. 2015; Brahmer et al. 2015]
VEGF	Breast, Cervical CRC, NSCLC RCC, Ovarian Glioblastoma	Bevacizumab	[Hurwitz et al. 2004; Sandler et al. 2006; Escudier et al. 2007; Giantonio et al. 2007; Miller et al. 2007; Friedman et al. 2009; Pujade-Lauraine et al. 2014; Tewari et al. 2014]
VEGF-R2	Gastric NSCLC	Ramucirumab	[Fuchs et al. 2014; Garon et al. 2014; Wilke et al. 2014]

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; BCR-ABL, breakpoint cluster region Abelson tyrosine kinase; CLL, chronic lymphocytic leukemia; CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; CRC, colorectal cancer; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; HER2, human epidermal growth factor receptor 2; NHL, non-Hodgkin’s lymphoma; NSCLC, non-small cell lung cancer; PAP, prostatic acid phosphatase; PD-1, programmed cell death receptor 1; RCC, renal cell carcinoma; VEGF, vascular endothelial growth factor; VEGF-R2, vascular endothelial growth factor receptor 2.

Immune checkpoint blockade: CTLA-4 and PD-1

A great deal of research in cancer immunotherapy is currently being concentrated on the development of immune checkpoint modulators, particularly those targeting CTLA-4 and PD-1 and its ligands PD-L1/L2. The first checkpoint modulator to be approved, ipilimumab is a monoclonal antibody directed against CTLA-4, a molecule that downregulates the activation of T cells. A phase III clinical trial in patients with metastatic melanoma comparing ipilimumab and gp100 peptide vaccine monotherapies to the combination showed that median OS was significantly increased from 6.4 months (gp100 vaccine alone) to 10.0 months (ipilimumab plus gp100 vaccine). Ipilimumab monotherapy was equally effective [Hodi et al. 2010]. The results of this trial, which was the first demonstration of an immunotherapeutic agent significantly increasing OS in patients with metastatic melanoma, led to the approval of ipilimumab in 2011. A subsequent phase III trial in patients with metastatic melanoma showed that a combination of ipilimumab and dacarbazine significantly increased OS (11.2 versus 9.1 months) compared with dacarbazine alone [Robert et al. 2011].

Still in clinical development, tremelimumab is another anti-CTLA-4 monoclonal antibody. Unlike ipilimumab, tremelimumab failed to demonstrate any significant clinical benefit in patients with advanced melanoma [Ribas et al. 2013], although a survival benefit was revealed in patients achieving higher drug exposures following a retrospective analysis of phase II and III pharmacokinetic data [Calabro et al. 2015]. Tremelimumab is currently being developed for the treatment of malignant mesothelioma [Calabro et al. 2015], where it has demonstrated long-term clinical benefit in phase II studies [Calabro et al. 2013, 2015]. The FDA recently granted tremelimumab Orphan Drug Designation for this indication.

Two new monoclonal antibody-based therapies targeting PD-1, pembrolizumab (formerly lambrolizumab) and nivolumab, were approved by the FDA within the last year. The interaction of PD-1, expressed on the surface of activated T cells, and PD-L1 on the surface of tumor cells results in immunosuppression. An expansion cohort of a phase I trial [Hamid et al. 2013] compared pembrolizumab treatment at doses of 2 or 10 mg in metastatic melanoma patients that had progressed on ipilimumab. At both dose levels, the overall response rate, which was the primary endpoint, was 26% and the treatment was well tolerated [Robert et al. 2014]. The results of this study led to accelerated FDA approval of pembrolizumab for metastatic melanoma in September 2014, representing the first such approval of an anti-PD-1 immunotherapy. Just 3 months later, nivolumab received accelerated FDA approval for advanced melanoma on the basis of phase I/II clinical trial data showing a 31% overall response rate with a median duration of response of two years, 43% two-year survival, and a median OS of approximately 17 months [Topalian et al. 2012, 2014]. A recently published phase III trial comparing a combination of nivolumab and dacarbazine to dacarbazine alone in patients with metastatic melanoma showed significant increases in progression-free survival (PFS) and OS with the combination [Robert et al. 2015a]. Clinical trials evaluating the use of nivolumab and pembrolizumab alone and in combination with other agents such as ipilimumab, TKIs, and anti-PD-L1 agents for the treatment of a wide variety of other cancers are currently ongoing or completed, with promising results [Scott, 2015; Swaika et al. 2015].

The results of clinical trials involving pembrolizumab and nivolumab in the treatment of non-small cell lung cancer (NSCLC) and advanced melanoma were recently reported. Pembrolizumab was found to be superior to ipilimumab in the treatment of patients with advanced melanoma in a phase III clinical trial [Robert et al. 2015b]. The 6-month PFS rate was 47.3% in patients treated with pembrolizumab compared with 26.5% in patients treated with ipilimumab, and the 12-month survival rate for pembrolizumab was 74.1% compared with 58.2% for ipilimumab, differences that were highly significant. A response rate of 33.7% was seen with pembrolizumab treatment compared with 11.9% for ipilimumab, with a lower incidence of adverse events in patients treated with pembrolizumab [Robert et al. 2015b]. In a phase I study in advanced NSCLC, pembrolizumab demonstrated antitumor activity, with a response rate of 45% among patients with a PD-L1 tumor cell expression rate of at least 50%, and a response rate of 19% among all patients. Median PFS was 3.7 months among all patients and 6.3 months in patients with high PD-L1 expression [Garon et al. 2015]. The results of this trial led to the recent FDA approval of pembrolizumab for NSCLC.

Nivolumab was also recently FDA approved for the treatment of NSCLC based on the results of a phase III study showing a significant increase in OS in patients treated with nivolumab compared with docetaxel [Brahmer et al. 2015]. In previously untreated patients with unresectable stage III/IV melanoma, a randomized, double-blind phase III trial showed that nivolumab treatment, either alone or in combination with ipilimumab, was superior to ipilimumab alone, with median PFS increasing significantly from 2.9 months with ipilimumab to 6.9 and 11.5 months with nivolumab and the combination, respectively. Among patients with PD-L1-positive tumors, median PFS was 14.0 months for nivolumab alone and the combination compared to 3.9 months for ipilimumab alone [Larkin et al. 2015]. The combination of nivolumab and ipilimumab recently received accelerated FDA approval for the treatment of BRAF V600 wild-type unresectable or metastatic melanoma based on the results of a phase II trial [Postow et al. 2015]. This was the first approval of an immunotherapy combination for cancer patients.

Investigational, PD-1-targeted agents still in clinical development include pidilizumab, which has been evaluated in phase II clinical trials in patients with B-cell lymphoma [Armand et al. 2013] and metastatic melanoma [Atkins et al. 2014], AMP-514 (MEDI0680), being evaluated in ongoing phase I trials in patients with advanced malignancies [ClinicalTrials.gov identifiers: NCT02118337; NCT02013804], and AMP-224, which is currently being evaluated in a phase I trial [ClinicalTrials.gov identifier: NCT02298946] in patients with metastatic colorectal cancer (CRC) [Homet Moreno et al. 2015].

EGFR receptor tyrosine kinase family

The human epidermal growth factor receptor (EGFR or HER1) is a receptor tyrosine kinase (RTK) that is overexpressed in a variety of different cancers including lung, breast and colon cancer. Specific mutations in the EGFR proto-oncogene result in the RTK becoming constitutively active [Chung, 2015]. Upon activation, EGFR stimulates a number of different signaling pathways, for example, MAPK (mitogen-activated protein kinase), PI3K (phosphoinositide 3-kinase) and STAT (signal transducer and activator of transcription), which lead to cancer cell proliferation and survival [Hynes and Lane, 2005]. This has made EGFR an attractive target for immunotherapy, and indeed several different monoclonal antibodies as well as small molecule TKIs have been developed and approved [Sliwkowski and Mellman, 2013]. Among these, gefitinib, erlotinib and afatinib are TKIs approved for the treatment of NSCLC. While TKIs are quite effective in treating EGFR mutation-positive NSCLC, the development of resistance is virtually universal, commonly through the T790M mutation [Pao et al. 2005]. Interestingly, a peptide antigen derived from the T790M EGFR mutation has been reported to be immunogenic and is a potential target for antigen-specific immunotherapy [Yamada et al. 2013; Ofuji et al. 2015].

Cetuximab and panitumumab

Approved monoclonal antibodies targeting EGFR include cetuximab for colon and head and neck cancers, and panitumumab for colon cancer. Cetuximab received accelerated FDA approval in 2004 for the treatment of metastatic CRC refractory to irinotecan based on the results of clinical trials showing acceptable safety [Saltz et al. 2004] and significant increases in overall response rate and time to progression in patients treated with a combination of cetuximab and irinotecan [Cunningham et al. 2004]. Phase III clinical trial data reaffirming prior results and demonstrating significant increases in PFS and OS in patients with refractory metastatic CRC [Jonker et al. 2007; Sobrero et al. 2008] led to regular FDA approval of single-agent cetuximab in 2007 for the treatment of chemotherapy-refractory metastatic CRC. More recently, cetuximab received FDA approval in 2012 as first-line treatment for metastatic CRC based on the results of the phase III CRYSTAL trial [Van Cutsem et al. 2009] and supporting trials [Bokemeyer et al. 2009, 2012]. A pooled analysis of these trials showed that cetuximab combined with chemotherapy significantly increased overall response rate, PFS and OS in patients with KRAS wild type, EGFR-expressing metastatic CRC [Bokemeyer et al. 2012].

Cetuximab is also used in the treatment of squamous cell carcinoma of the head and neck (SCCHN), having received FDA approval in 2006 for single-agent use in recurrent/metastatic SCCHN refractory to platinum-based chemotherapy, and in combination with radiotherapy for locally or regionally advanced SCCHN. This approval was based on the results of a phase III clinical trial showing significant increases in PFS, median duration of locoregional control and OS with cetuximab plus radiotherapy compared to radiotherapy alone in patients with locoregionally advanced SCCHN [Bonner et al. 2006], and a phase II study in patients with refractory recurrent/metastatic SCCHN showing a 13% response rate and median OS of approximately 6 months with cetuximab monotherapy [Vermorken et al. 2007]. Cetuximab was subsequently approved as first-line therapy in combination with chemotherapy for recurrent/metastatic SCCHN on the strength of the results of a phase III clinical trial showing significant increases in median OS, PFS and response rate with the combination of cetuximab and chemotherapy compared with chemotherapy alone in patients with recurrent/metastatic SCCHN [Vermorken et al. 2008]. The activity of cetuximab has also been evaluated in NSCLC, where a meta-analysis of four phase II/III clinical trials showed that the combination of cetuximab and first-line chemotherapy slightly, yet significantly, increased OS compared with platinum-based chemotherapy alone [Pujol et al. 2014].

Panitumumab is also used in the treatment of metastatic CRC. The results of a phase III clinical trial showing significant increases in PFS and response rate in patients with chemotherapy-refractory metastatic CRC led to the accelerated FDA approval of panitumumab in 2006 [Van Cutsem et al. 2007]. Panitumumab was most recently approved in 2014 as first-line treatment in combination with chemotherapy in KRAS wild-type, metastatic CRC following the results of phase III clinical trials showing a significant increase in PFS, an increase in OS [Douillard et al. 2010], and noninferiority with cetuximab [Price et al. 2014].

Trastuzumab

Trastuzumab is a monoclonal antibody used for the treatment of breast cancer overexpressing the human EGFR 2 (HER2) oncogene, which includes approximately 15–30% of breast cancers and is associated with a poor prognosis [Slamon et al. 1987, 1989]. The FDA first approved trastuzumab in combination with chemotherapy in 1998 for the first-line treatment of patients with metastatic breast cancer. This original approval was based on the results of a phase III clinical trial showing that treatment with trastuzumab plus standard chemotherapy significantly increased time to progression, response rate, duration of response and OS compared with chemotherapy alone [Slamon et al. 2001]. Based on clinical trials data showing significant increases in disease-free survival (DFS) and OS [Romond et al. 2005; Perez et al. 2011, 2014], trastuzumab later received FDA approval in 2006 for the treatment of breast cancer in the adjuvant setting in combination with chemotherapy. Trastuzumab was most recently approved in 2010 for the first-line treatment of metastatic gastric or gastro-esophageal junction adenocarcinoma in combination with chemotherapy based on a phase III clinical trial showing significantly increased OS following treatment with trastuzumab combined with chemotherapy [Bang et al. 2010]. Currently, trastuzumab in combination with pembrolizumab is being evaluated in a phase I/II clinical trial in patients with trastuzumab-resistant, HER2-positive metastatic breast cancer [ClinicalTrials.gov identifier: NCT02129556]. It has been previously shown that trastuzumab and anti-PD-1 treatment are synergistic in mice [Stagg et al. 2011].

Necitumumab

Still in clinical development, necitumumab is a second-generation anti-EGFR monoclonal antibody currently being evaluated for the treatment of NSCLC. Recently published data from a phase III clinical trial in 1093 patients with stage IV squamous NSCLC showed that necitumumab in combination with chemotherapy as first-line treatment resulted in a modest survival benefit, with a median OS of 11.5 months in patients treated with necitumumab plus chemotherapy compared with 9.9 months in patients treated with chemotherapy alone, a difference that was significant [Thatcher et al. 2015]. In patients with stage IV nonsquamous NSCLC, however, combining necitumumab with standard chemotherapy as first-line treatment provided no additional clinical benefit compared with chemotherapy alone [Paz-Ares et al. 2015].

VEGFA and VEGF-R2

Vascular endothelial growth factor A (VEGFA) plays a critical role in angiogenesis, and it is the target of the monoclonal antibody bevacizumab. In addition to its anti-angiogenic effects [Ferrara et al. 2004], bevacizumab has been found to augment tumor infiltration by T and B lymphocytes [Manzoni et al. 2010; Hodi et al. 2014] and to inhibit regulatory T cells (Tregs) [Terme et al. 2013]. Bevacizumab has been FDA approved for the treatment of CRC (2004), glioblastoma (2009), cervical cancer (2014), lung cancer (2006), renal cell cancer (2009), breast cancer (2008), and ovarian cancer (2014).

Bevacizumab was initially approved for the first-line treatment of metastatic CRC in combination with chemotherapy based on a phase III study showing significant increases in PFS and median OS compared with chemotherapy alone [Hurwitz et al. 2004]. Shortly thereafter, bevacizumab received approval as second-line treatment in combination with chemotherapy for patients with previously treated metastatic CRC following the results of a phase III clinical trial showing significant improvement in PFS and median OS [Giantonio et al. 2007] compared with chemotherapy alone. Two years later, another Phase III trial in patients with recurrent or advanced NSCLC showed significant increases in response rate, PFS and OS following treatment with bevacizumab plus chemotherapy [Sandler et al. 2006], which led to approval of bevacizumab combined with chemotherapy as first-line treatment of recurrent/advanced NSCLC. The approval of bevacizumab as first-line treatment of metastatic breast cancer was supported by the results of a phase III clinical trial in patients with HER2-negative metastatic breast cancer showing that treatment with bevacizumab and paclitaxel led to significant increases in PFS and objective response rate (ORR) compared with paclitaxel alone [Miller et al. 2007]. A phase II study used to support the 2009 FDA approval of bevacizumab combined with chemotherapy for patients with recurrent glioblastoma showed that treatment with bevacizumab combined with chemotherapy resulted in improved PFS and ORR, differences that were significant compared to expected effects of salvage chemotherapy [Friedman et al. 2009]. The same year, bevacizumab combined with interferon α was approved as first-line treatment of metastatic renal cell carcinoma (RCC) based on phase III data demonstrating significant improvement in PFS [Escudier et al. 2007]. No significant improvement in OS was observed in this trial [Escudier et al. 2010]. Most recently, bevacizumab was approved in 2014 for the treatment of recurrent or metastatic cervical cancer and recurrent, platinum-refractory ovarian carcinoma based on phase III clinical trials showing significant increases in OS and response rate in cervical cancer [Tewari et al. 2014], and significant improvement in PFS and ORR in ovarian cancer [Pujade-Lauraine et al. 2014], compared with chemotherapy alone. Like trastuzumab, bevacizumab and anti-PD-1 combination treatment is currently being clinically evaluated [ClinicalTrials.gov identifiers: NCT02210117; NCT02039674; NCT02348008].

VEGF-R2, also known as KDR (kinase insert domain receptor) and Flk-1 (fetal liver kinase 1), is an RTK and the primary receptor through which VEGF mediates its angiogenic, mitogenic, and permeability-enhancing effects [Ferrara et al. 2004]. A monoclonal antibody that targets VEGF-R2, ramucirumab has received FDA approval for the second-line treatment of gastric cancer and NSCLC within the past year. Ramucirumab was approved as monotherapy and in combination with paclitaxel for the treatment of gastric or gastroesophageal junction adenocarcinoma after progression on first-line chemotherapy based on the results of two phase III trials showing significant increases in median OS compared with placebo [Fuchs et al. 2014] and paclitaxel plus placebo [Wilke et al. 2014]. Efficacy as second-line treatment for metastatic NSCLC after progression on platinum-based chemotherapy was also demonstrated in a phase III trial showing significant improvement in PFS and OS with the combination of docetaxel and ramucirumab compared with docetaxel plus placebo [Garon et al. 2014].

PAP

PAP is the target of the only currently FDA-approved therapeutic anticancer vaccine, sipuleucel-T (Provenge^®). Sipuleucel-T was approved for the treatment of asymptomatic and minimally symptomatic metastatic castration-resistant prostate cancer in 2010 based on the results of the phase III IMPACT trial showing a significant increase in median OS in patients treated with sipuleucel-T compared with those treated with placebo [Kantoff et al. 2010a]. To prepare sipuleucel-T, peripheral blood mononuclear cells from the patient are cultured with a fusion protein that incorporates PAP and GM-CSF (granulocyte-macrophage colony-stimulating factor). The patient is then infused with these cells, and the process is repeated for three cycles. Thus, sipuleucel-T is actually a combination of a cell-based vaccine and autologous ACT [Melero et al. 2014]. Interestingly, in addition to immune responses against the primary antigenic target PAP, humoral immune responses against several secondary antigens such as PSA (prostate specific antigen), K-ras and KLK2/hK2 were also observed to be elevated in patients treated with sipuleucel-T but not control in the phase III IMPACT trial, and these responses were associated with improved OS [GuhaThakurta et al. 2015].

Investigational tumor antigens and their clinical potential

In addition to the established tumor antigens for which targeted immunotherapies have already been approved (Table 1), there are numerous novel tumor antigens currently being investigated as potential targets for new immunotherapies, including MUC1, PD-L1, lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), V-domain immunoglobulin-containing suppressor of T-cell activation (VISTA), NY-ESO-1, carcinoembryonic antigen (CEA), PSA, MAGE-A3, 5T4, survivin and indoleamine-2,3-dioxygenase (IDO1). These selected antigens and their clinical potential as targets for investigational immunotherapies are discussed below. Table 2 includes a more complete listing of tumor antigen targets for which immunotherapies are currently being explored.

Table 2.

Antigenic targets of investigational immunotherapies.

Antigen	Cancer types	Targeted agents	References
A2aR	NSCLC	PBF-509	[Pinna, 2014]
AKAP4	NSCLC Ovarian	Preclinical	[Agarwal et al. 2013; Mirandola et al. 2015]
BAGE	Glioblastoma Ovarian	Preclinical	[Zhang et al. 2010; Akiyama et al. 2014]
BORIS	Prostate, Lung Esophageal	Preclinical	[Okabayashi et al. 2012; Cheema et al. 2014; Lee et al. 2014]
CD22	ALL	Epratuzumab Moxetumomab Inotuzumab ozogamicin	[Raetz et al. 2008; Advani et al. 2014; Annesley and Brown, 2015; Raetz et al. 2015]
CD73	Advanced solid tumors	MEDI9447	[Hay et al. 2015]
CD137	Advanced solid tumors	Urelumab PF-05082566	[Yonezawa et al. 2015]
CEA	CRC	PANVAC™ Ad5-[E1-, E2b-]-CEA(6D)	[Turriziani et al. 2012; Morse et al. 2013a, 2013b]
CS1	Multiple myeloma	Elotuzumab	[Veillette and Guo, 2013]
CTLA-4	Malignant mesothelioma	Tremelimumab	[Calabro et al. 2015]
EBAG9	Bladder	Preclinical	[Miyazaki et al. 2014]
EGF	NSCLC	CIMAvax	[Gonzalez et al. 2007; Neninger Vinageras et al. 2008; Cheng and Kananathan, 2012; Ruiz et al. 2014]
EGFR	NSCLC	Necitumumab	[Thatcher et al. 2015]
GAGE	Cervical	Preclinical	[Kular et al. 2010]
GD2	Neuroblastoma Retinoblastoma Melanoma other solid tumors	Dinutuximab, hu3F8 hu14.18-IL-2, 3F8/OKT3BsAb anti-GD2 CAR GD2-KLH	[Suzuki and Cheung, 2015]
gp100	Melanoma	gp100:209-217(210M)	[Schwartzentruber et al. 2011]
HPV-16	Cervical SCCHN	HPV-16 (E6, E7) TG4001, Lm-LLO-E7 pNGVL4a-CRT/E7, INO-3112	[Kenter et al. 2009; Brun et al. 2011; Bagarazzi et al. 2012; Zaravinos, 2014]
HSP105	CRC Bladder	Preclinical	[Zappasodi et al. 2011; Kawai et al. 2014; Sawada et al. 2014]
IDH1	Glioma	IDH1(R132H) p123-142	[Schumacher et al. 2014]
Idiotype (NeuGcGM3)	NSCLC, Breast Melanoma	Racotumomab	[Alfonso et al. 2002, 2014; Diaz et al. 2003]
IDO1	Breast, Melanoma NSCLC	Indoximod INCB024360 IDO1 peptide vaccine	[Iversen et al. 2014, 2015; Soliman et al. 2014]
KIR	Lymphoma	Lirilumab	[Kohrt et al. 2014]
LAG-3	Breast, Hemato- logical, Advanced solid tumors	BMS-986016 IMP321	[Brignone et al. 2010; Creelan, 2014; Nguyen and Ohashi, 2015]
LY6K	Gastric SCCHN	LY6K-177 peptide LY6K, CDCA1, IMP3	[Ishikawa et al. 2014; Yoshitake et al. 2015]
MAGE-A3	Melanoma NSCLC	recMAGE-A3 Zastumotide	[Kruit et al. 2013; Ulloa-Montoya et al. 2013; Vansteenkiste et al. 2013; Melero et al. 2014]
MAGE-C2	Gastric, Melanoma Multiple myeloma	Preclinical	[Zhang et al. 2013; Ghadban et al. 2014; Reinhard et al. 2014]
MAGE-D4	CRC	Preclinical	[Zhang et al. 2014]
Melan-A	Melanoma	MART-1 (26-35, 27L)	[Tarhini et al. 2012; Romano et al. 2014]
MET	NSCLC	Onartuzumab Tivantinib	[Scagliotti et al. 2012; Spigel et al. 2013; Stinchcombe, 2014]
MUC1	NSCLC, Breast Prostate	Tecemotide, TG4010 PANVAC™	[Kimura and Finn, 2013; Butts et al. 2014; Wurz et al. 2014]
MUC4	Pancreatic	Preclinical	[Wu et al. 2009; Torres et al. 2012; Zhu et al. 2014]
MUC16	Ovarian	Abagovomab Oregovomab	[Buzzonetti et al. 2014; Felder et al. 2014]
NY-ESO-1	Ovarian Melanoma	NY-ESO-1/ISCOMATRIX™ rV-NY-ESO-1; rF-NY-ESO-1	[Robbins et al. 2011; Odunsi et al. 2012; Klein et al. 2015]
PD-1	B-cell lymphoma Melanoma, CRC	Pidilizumab AMP-224, AMP-514	[Armand et al. 2013; Homet Moreno et al. 2015]
PD-L1	NSCLC, RCC Bladder, Breast Melanoma, SCCHN	BMS-936559, Atezolizumab Durvalumab, Avelumab	[Cha et al. 2015; Ibrahim et al. 2015; Sunshine and Taube, 2015; Swaika et al. 2015]
PRAME	NSCLC	Preclinical	[De Pas et al. 2012]
PSA	Prostate	PROSTVAC®-VF	[Gulley et al. 2010; Kantoff et al. 2010b; Shore, 2014]
ROR1	CLL, Pancreatic Lung, Breast	Preclinical	[Hojjat-Farsangi et al. 2014; Berger et al. 2015; Shabani et al. 2015]
Sialyl-Tn	Breast	Theratope	[Miles et al. 2011; Ibrahim et al. 2013]
SPAG-9	Prostate, CRC NSCLC, Ovarian	Preclinical	[Garg et al. 2007; Kanojia et al. 2011; Wang et al. 2013; Chen et al. 2014]
SSX1	Prostate Multiple myeloma	Preclinical	[Smith et al. 2011; He et al. 2014]
Survivin	Melanoma Glioma, Solid tumors	EMD640744 Trivalent peptide vaccine Tripeptide vaccine	[Becker et al. 2012; Lennerz et al. 2014; Pollack et al. 2014]
Telomerase	Pancreatic	Tertomotide	[Melero et al. 2014; Staff et al. 2014]
TIM-3	Melanoma, NHL NSCLC	Preclinical	[Sakuishi et al. 2010; Ngiow et al. 2011; Gao et al. 2012; Fourcade et al. 2014; Jiang et al. 2015]
VISTA	Melanoma, Bladder	Preclinical	[Le Mercier et al. 2014; Lines et al. 2014b]
WT1	Ovarian, Uterine, AML Multiple myeloma	WT1 peptide vaccine	[Coosemans et al. 2014; Di Stasi et al. 2015]
XAGE-1b	Prostate	DC-based tumor vaccine	[Zhou et al. 2008; Xie and Wang, 2015]
5T4	RCC, CRC Prostate	TroVax® Naptumomab estafenatox	[Amato et al. 2010; Zhang et al. 2012; Harrop et al. 2013; Eisen et al. 2014; Rowe and Cen, 2014; Stern et al. 2014]

Abbreviations: A2aR, adenosine A2a receptor; AKAP4, A kinase anchor protein 4; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; BAGE, B melanoma antigen; BORIS, brother of the regulator of imprinted sites; CEA, carcinoembryonic antigen; CLL, chronic lymphocytic leukemia; CRC, colorectal cancer; CS1, CD2 subset 1; CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; EBAG9, estrogen receptor binding site associated antigen 9; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; GAGE, G antigen; GD2, disialoganglioside GD2; gp100, glycoprotein 100; HPV-16, human papillomavirus 16; HSP105, heat-shock protein 105; IDH1, isocitrate dehydrogenase type 1; IDO1, indoleamine-2,3-dioxygenase 1; KIR, killer cell immunoglobulin-like receptor; LAG-3, lymphocyte activation gene 3; LY6K, lymphocyte antigen 6 complex K; MAGE-A3, melanoma antigen 3; MAGE-C2, melanoma antigen C2; MAGE-D4, melanoma antigen D4; Melan-A/MART-1, melanoma antigen recognized by T-cells 1; MET, N-methyl-N’-nitroso-guanidine human osteosarcoma transforming gene; MUC1, mucin 1; MUC4, mucin 4; MUC16, mucin 16; NHL, non-Hodgkin lymphoma; NY-ESO-1, New York esophageal squamous cell carcinoma 1; PD-1, programmed cell death receptor 1; PD-L1, programmed cell death receptor ligand 1; PRAME, preferentially expressed antigen of melanoma; PSA, prostate specific antigen; RCC, renal cell carcinoma; ROR1, receptor tyrosine kinase orphan receptor 1; SCCHN, squamous cell carcinoma of the head and neck; SPAG-9, sperm-associated antigen 9; SSX1, synovial sarcoma X-chromosome breakpoint 1; TIM-3, T-cell immunoglobulin domain and mucin domain-3; VISTA, V-domain immunoglobulin-containing suppressor of T-cell activation; WT1, Wilms’ Tumor-1; XAGE-1b, X chromosome antigen 1b.

MUC1

Mucin 1 (MUC1) is one of the best-characterized TAAs, and it is overexpressed and aberrantly glycosylated in over 90% of adenocarcinomas such as lung and breast cancer [Szabo, 2003; Brayman et al. 2004]. MUC1 is a transmembrane glycoprotein expressed in several epithelial tissues, but the normal glycosylation pattern shields the peptide core of the extracellular domain from immunosurveillance. In cancer, the aberrant underglycosylation of MUC1 leads to exposure of the immunogenic tandem repeat regions of the core peptide, making MUC1 an attractive target for immunotherapies [Apostolopoulos and McKenzie, 1994; Vlad et al. 2004].

While several different MUC1-targeted immunotherapies have been assessed in clinical trials [Kimura and Finn, 2013], the most advanced agent is tecemotide, an antigen-specific, liposome-based, peptide cancer vaccine [Wurz et al. 2014] that has been evaluated in a phase III clinical trial as maintenance therapy for patients with locally advanced, unresectable stage IIIA/IIIB NSCLC who had not progressed following primary chemoradiotherapy [Butts et al. 2014]. The results failed to demonstrate significant improvement in OS with tecemotide compared with placebo; however, a predefined subgroup analysis of the patients who received concurrent chemoradiotherapy followed by tecemotide showed a significant survival benefit, while no survival benefit was seen with sequential chemoradiotherapy [Butts et al. 2014]. After approximately 20 months of additional follow up, the survival benefit of tecemotide following concurrent chemoradiotherapy remained significant [Mitchell et al. 2015]. These results suggest that the timing of radiotherapy, chemotherapy and immunotherapy is important and that monitoring immune status may be necessary when designing treatment regimens combining immunotherapy with chemoradiotherapy [Kao et al. 2014, 2015]. Another phase III trial, recently discontinued, was initiated to study tecemotide maintenance therapy following concurrent chemoradiotherapy in NSCLC [DeGregorio et al. 2014].

Immune checkpoints: PD-L1; LAG-3; TIM-3; VISTA

PD-L1

As already mentioned, the development of immune checkpoint inhibitors is currently the focus of intense research efforts. The interaction of PD-L1, which is expressed on the surface of tumor cells, with PD-1 on activated T cells results in immunosuppression and tumor immune escape [Iwai et al. 2002; Swaika et al. 2015]. PD-L1 expression has been found to be inducible by interferon γ [Muhlbauer et al. 2006]. Several investigational immunotherapies targeting PD-L1 are presently in clinical development for metastatic melanoma, NSCLC, RCC and bladder cancer, among others.

BMS-936559 (MDX-1105) is a monoclonal antibody that inhibits the binding of PD-L1 to both PD-1 and CD80. The safety and antitumor activity of BMS-936559 in patients with a variety of advanced cancers have been evaluated in a phase I clinical trial involving 207 patients [Brahmer et al. 2012]. In NSCLC, an ORR of 10% (all partial responses) and PFS rate of 31% at 24 weeks were observed. Patients with RCC showed a 12% ORR and 53% progression-free response rate at 24 weeks. In ovarian cancer, an ORR of only 6% was observed [Brahmer et al. 2012]. No more recent clinical data regarding BMS-936559 in cancer therapy have emerged, and it appears that clinical development of this agent for oncology applications has been discontinued [Mullard, 2013; Sunshine and Taube, 2015].

Atezolizumab (MPDL3280A) is an anti-PD-L1 monoclonal antibody that has demonstrated activity against a variety of advanced cancers in phase I clinical trials. The Fc region of the antibody is modified to prevent both ADCC and complement-dependent cytotoxicity (CDC) [Lu et al. 2014], which prevents the killing of immune cells that also express PD-L1, such as activated T cells. Atezolizumab is currently being evaluated in patients with locally advanced or metastatic solid tumors in an ongoing phase I study. In a cohort of patients with metastatic urothelial bladder cancer, an ORR of 43% was observed at 6 weeks, increasing to 52% at 12 weeks of treatment [Powles et al. 2014]. As there had been no significant advances in the treatment of metastatic urothelial cancer in decades, these results led to breakthrough designation status by the FDA in June 2014. Updated response and survival data from this study after an additional 8 months of follow up continued to show promising PFS and OS results [Petrylak et al. 2015]. For patients with NSCLC, an ORR of 23% and PFS rate of 45% at 24 weeks was seen with atezolizumab treatment [Herbst et al. 2014], and the most recent data continued to show clinical benefit in NSCLC [Horn et al. 2015]. Atezolizumab has also been studied in combination with first-line chemotherapy in metastatic NSCLC patients, and was found to improve ORR in all combinations tested in a phase Ib trial [Liu et al. 2015b]. In patients with metastatic melanoma or RCC, ORRs of 30% and 14%, and PFS rates of 41% and 48% at 24 weeks, respectively, were observed [Herbst et al. 2014]. Patients in the triple-negative breast cancer cohort had an ORR of 19% and a PFS rate of 27% at 24 weeks [Emens et al. 2015].

Preliminary results from two phase II clinical trials of atezolizumab in patients with advanced NSCLC were recently reported. In both chemotherapy-naïve and previously treated patients, an increasing ORR to atezolizumab treatment was associated with increasing PD-L1 expression [Spigel et al. 2015]. Compared with docetaxel in previously treated NSCLC, treatment with atezolizumab was found to increase OS, PFS and ORR in association with PD-L1 expression [Spira et al. 2015]. There are currently several other phase I/II and phase III clinical trials of atezolizumab currently underway [Swaika et al. 2015].

Durvalumab (MEDI4736) is an anti-PD-L1 monoclonal antibody similar to atezolizumab, with an Fc region modification that prevents ADCC and CDC. In a phase I clinical trial, response rates of 13% and 14% were observed in patients with metastatic NSCLC and SCCHN, respectively [Lee and Chow, 2014]. A phase II study [ClinicalTrials.gov identifier: NCT02336165] in patients with glioblastoma multiforme is currently underway [Reardon et al. 2015]. Durvalumab has also entered a phase III clinical trial in combination with concurrent chemoradiotherapy in patients with stage III unresectable NSCLC [ClinicalTrials.gov identifier: NCT02125461] and a phase II/III clinical trial [ClinicalTrials.gov identifier: NCT02154490] in patients with recurrent stage IIIB/IV squamous cell NSCLC [Lee and Chow, 2014; Ibrahim et al. 2015]. Other ongoing phase II and phase III clinical trials of durvalumab include third-line treatment of stage IIIB/IV NSCLC [ClinicalTrials.gov identifier: NCT02087423], second-line treatment of SCCHN [ClinicalTrials.gov identifier: NCT02207530; Ibrahim et al. 2015] and treatment of advanced NSCLC alone or in combination with tremelimumab [ClinicalTrials.gov identifier: NCT02352948; Planchard et al. 2015].

A fourth anti-PD-L1 monoclonal antibody, avelumab (MSB0010718C), is currently in phase I development in subjects with metastatic or locally advanced solid tumors [ClinicalTrials.gov identifier: NCT01772004], with a separate phase I trial in Japanese subjects [Lu et al. 2014]. Results of this phase I trial in extensively pretreated patients with a variety of different tumors have so far demonstrated that avelumab has an acceptable safety profile [Kelly et al. 2015]. A phase Ib expansion study in advanced NSCLC patients who had progressed after platinum-based chemotherapy demonstrated increased activity with avelumab treatment in patients with PD-L1-positive tumors [Gulley et al. 2015]. A phase II study of avelumab in patients with metastatic Merkel cell carcinoma is currently underway [Kaufman et al. 2015]. Interestingly, unlike the other anti-PD-L1 antibodies being developed, avelumab appears to possess ADCC activity [Boyerinas et al. 2015].

LAG-3

LAG-3 is a negative regulator of effector T-cell function [Sierro et al. 2011], making it an attractive target for immunotherapy. LAG-3 is expressed on the membranes of activated T cells and is upregulated on exhausted T cells [Nguyen and Ohashi, 2015]. Because LAG-3 is also expressed on activated Tregs, LAG-3 blockade may inhibit the suppressive effects of Tregs in addition to the direct action on effector cells [Huang et al. 2004]. The major ligand of LAG-3 is MHC class II, and their interaction may play a role in modulating dendritic cell function [Goldberg and Drake, 2011; Camisaschi et al. 2014]. A monoclonal antibody targeting LAG-3, known as BMS-986016, is currently in early phase I clinical development. Ongoing clinical trials are investigating single agent BMS-986016 for hematological malignancies and a combination of nivolumab and BMS-986016 in patients with advanced solid tumors [Creelan, 2014; Nguyen and Ohashi, 2015]. In addition to the membrane-bound form of LAG-3, the LAG3 gene also codes for a soluble variant, sLAG-3, that functions as an immune adjuvant [Nguyen and Ohashi, 2015]. A sLAG-3 immunoglobulin fusion protein (IMP321) is in early clinical development as an immune adjuvant. In a Phase I clinical trial evaluating a combination of taxane-based chemotherapy and IMP321 in women with metastatic breast cancer, a 50% ORR was observed [Brignone et al. 2010].

TIM-3

TIM-3 is an inhibitory immune checkpoint receptor that limits the duration and magnitude of T_H1 CD4+ and CD8+ CTL cellular responses [Anderson, 2014]. Galectin-9 has been identified as the ligand for TIM-3, and it is through this pathway that T_H1 immunity may be suppressed [Zhu et al. 2005]. The expression of TIM-3 on dysfunctional CD8+ T cells and Tregs makes it an attractive target for anticancer immunotherapy. Preclinical animal models have shown that antibody inhibition of TIM-3 is very effective in suppressing tumor growth [Ngiow et al. 2011]. Synergistic antitumor effects in multiple cancer types have been observed in preclinical models with the combination of anti-PD-1/PD-L1 and TIM-3 blockade [Sakuishi et al. 2010; Ngiow et al. 2011; Jing et al. 2015]. Clinically, increased expression of TIM-3 and PD-1 in melanoma patients has been associated with tumor NY-ESO-1-specific CD8+ T-cell dysfunction [Fourcade et al. 2014]. In NSCLC, TIM-3 is expressed on tumor-infiltrating CD4+ and CD8+ T cells [Gao et al. 2012], and the presence of CD4+ T cells that express TIM-3 is strongly correlated with advanced tumor grade in NSCLC [Gao et al. 2012] and poor survival in follicular B-cell non-Hodgkin lymphoma [Yang et al. 2012]. Thus, TIM-3 has great clinical potential as a target of novel immunotherapies [Cheng and Ruan, 2015].

VISTA

VISTA is a newly identified negative immune checkpoint regulator [Lines et al. 2014b]. Unlike PD-L1, VISTA is expressed primarily in hematopoietic cells in both mice [Wang et al. 2011] and humans [Lines et al. 2014a]. In murine models of melanoma and bladder cancer, leukocytes within the tumor microenvironment and in tumor-draining lymph nodes were found to have elevated VISTA expression. Treatment with an anti-VISTA monoclonal antibody resulted in tumor growth inhibition, especially in combination with a tumor vaccine [Le Mercier et al. 2014]. In these studies, VISTA blockade was found to be effective with no detectable expression of VISTA and high expression levels of PD-L1 on tumor cells. This observation could have important clinical implications for combination treatments targeting the VISTA and PD-1/PD-L1 checkpoint pathways, allowing for potential synergy due to the fact that these pathways operate independently [Lines et al. 2014b; Liu et al. 2015a].

CTAs: NY-ESO-1 and MAGE-A3

CTAs constitute the majority of the currently identified TAAs, and numerous CTAs are being investigated as potential targets of new anticancer immunotherapies for a variety of malignancies. Among these, immunotherapies targeting NY-ESO-1 and MAGE-A3 have reached the clinical stage of development. These antigens are normally expressed only in adult testis germ cells and become aberrantly re-expressed in various cancers such as lung, bladder and melanoma [Jungbluth et al. 2001].

NY-ESO-1

NY-ESO-1 is encoded by the CTAG1B gene and was first identified in 1997 [Chen et al. 1997]. Expressed in a variety of carcinomas and sarcomas, NY-ESO-1 is highly immunogenic and is thus a logical target for anticancer immunotherapy [Nicholaou et al. 2006; Endo et al. 2015]. In a phase I clinical trial in patients with advanced synovial cell sarcoma and melanoma, subjects were administered autologous ACT using cells transduced with a T-cell receptor (TCR) targeting NY-ESO-1. Objective response rates of 67% (4/6) and 45% (5/11) were observed for synovial cell sarcoma and melanoma patients, respectively [Robbins et al. 2011]. Autologous ACT has also been performed using NY-ESO-1-specific CD4+ cells [Hunder et al. 2008]. A prime-boost vaccine strategy was assessed in patients with stage III/IV melanoma and stage II-IV ovarian carcinoma in a phase II clinical trial. Patients were primed with a recombinant NY-ESO-1-expressing vaccinia virus and then boosted with an NY-ESO-1-expressing fowlpox virus. In the melanoma patients, CD8+ T-cell responses increased from 40% pretreatment to 88% following vaccination. Median OS was significantly increased in the melanoma and ovarian cancer patients who had immune responses following treatment [Odunsi et al. 2012]. An NY-ESO-1-specific vaccine (NY-ESO-1/ISCOMATRIX™) in combination with cyclophosphamide has also been evaluated in advanced melanoma in phase II. Treatment led to a significant increase in antigen-specific CD4+ T-cell immune responses [Klein et al. 2015] compared to a cohort of patients treated with the vaccine alone [Nicholaou et al. 2009]. A prime-boost treatment strategy employing NY-ESO-1/ISCOMATRIX™ in high-risk, resected melanoma patients was found to augment CD8+ T-cell immune responses [Chen et al. 2015].

MAGE-A3

MAGE-A3 is a CTA that has been extensively clinically investigated as a target for immunotherapy in melanoma and lung cancer. In a clinical trial involving nine patients with advanced cancers, five responses were observed following autologous ACT with T cells transduced with anti-MAGE-A3 TCRs. However, neurological toxicities were seen in three patients, resulting in two deaths due to previously unknown expression of MAGE family antigens in the brain [Morgan et al. 2013]. A Phase II study examined vaccination with recombinant MAGE-A3 protein combined with either AS15 [a combination of QS21 saponin, monophosphoryl lipid A (MPL-A), and CpG7909, a Toll-like receptor (TLR)-9 agonist, in a liposomal formulation] or AS02_B (a combination of QS21 saponin and MPL-A, a TLR-4 agonist) immunostimulants in patients with stage III/IV melanoma. Trends towards greater ORR, PFS and median OS were observed in the patients who received the AS15 immunostimulant compared with AS02_B [Kruit et al. 2013]. The peptide vaccine zastumotide (GSK2132231A) is being evaluated in a phase III clinical trial in patients with resected stage IIIB/IIIC melanoma. This trial failed to demonstrate a significant prolongation of DFS in patients with positive expression of MAGE-A3 [Melero et al. 2014], but it is ongoing in a subset of patients who are positive for a predictive marker of response [Ulloa-Montoya et al. 2013]. A phase II clinical trial of zastumotide (GSK1572932A) in patients with resected stage IB/II NSCLC exhibited a trend toward positive treatment outcomes compared with placebo [Vansteenkiste et al. 2013]; however, a large phase III study in patients with resected stage IB-IIIA NSCLC failed to meet its primary endpoint of significant prolongation of DFS, resulting in termination of the study [Melero et al. 2014]. Clinical development of zastumotide appears to have been discontinued.

Oncofetal antigens: CEA and 5T4

Oncofetal antigens are so-called because though widely expressed during fetal development, normal expression is generally limited in adults. In cancer, the expression of these antigens becomes upregulated, making them potential targets for immunotherapeutic intervention.

CEA

CEA is a well-known oncofetal antigen normally expressed in the stomach, tongue, esophagus, cervix and prostate in adults, but becomes highly overexpressed in colorectal and gastric cancers [Beauchemin and Arabzadeh, 2013]. Clinically, CEA has been investigated extensively as an immunotherapeutic target for colorectal and various other cancers [Turriziani et al. 2012]. In a phase I/II clinical trial, treatment with an adenoviral gene delivery platform encoding the CEA antigen produced cell-mediated immunity in 61% of patients with advanced CRC and a 48% OS rate at 12 months [Morse et al. 2013a]. A phase II study in CRC patients who were disease-free following metastasectomy and perioperative chemotherapy compared the effectiveness of autologous dendritic cells modified with PANVAC™, a poxvector encoding both CEA and MUC1, to the PANVAC™ poxvector administered in combination with GM-CSF. Survival and recurrence-free survival were similar between the two treatment groups, and vaccinated patients overall experienced superior survival compared with a contemporary group of patients not treated with immunotherapy [Morse et al. 2013b]. In a recently reported phase II trial comparing treatment with PANVAC™ in combination with docetaxel to docetaxel alone in 48 patients with metastatic breast cancer, patients treated with the combination experienced improved PFS compared with docetaxel alone, a result which, while not significant, was suggestive of a clinical benefit [Heery et al. 2015]. Increased CEA expression has been associated with shorter relapse-free survival in breast cancer patients [Saadatmand et al. 2013].

5T4

The oncofetal antigen 5T4 was identified by screening for shared surface molecules in human trophoblasts and human cancer cells [Stern et al. 2014]. A heavily glycosylated, membrane-bound protein, 5T4 is highly expressed in cervical, colorectal, gastric, ovarian, prostate, lung and renal cancers [Southall et al. 1990]. The expression of 5T4 has been associated with epithelial mesenchymal transition (EMT), which is involved in the metastasis of epithelial cancers [Nieto and Cano, 2012]. Three different 5T4-based immunotherapeutic strategies are being clinically evaluated: a vaccine known as TroVax^® (modified vaccinia virus Ankara- MVA), an antibody–superantigen [Staphylococcal Enterotoxin A (SEA)] fusion protein, and an antibody–drug conjugate (ADC) combining a 5T4-specific monoclonal antibody with a tubulin inhibitor.

5T4 vaccine (TroVax®)

Favorable trends toward clinical benefit have been observed in early phase I and phase II clinical trials of the TroVax^® 5T4-MVA vaccine in advanced RCC [Zhang et al. 2012], CRC [Rowe and Cen, 2014], and castration-resistant prostate cancer [Harrop et al. 2013]. To determine whether treatment with TroVax^® can improve survival, a phase III clinical trial in patients with metastatic RCC compared treatment with TroVax^® to standard of care treatment [Amato et al. 2010]. While this trial failed to demonstrate any significant effects of TroVax^® on OS, a subgroup of patients with good prognosis and receiving concurrent IL-2 experienced a significant survival benefit compared with placebo. The patients with the greatest increases in 5T4-specific antibodies also experienced a survival benefit with TroVax^® compared with placebo, which is consistent with a pooled analysis of prior phase I/II TroVax^® studies [Harrop et al. 2010].

5T4 superantigen–antibody fusion protein (naptumomab estafenatox)

The tumor-targeted super-antigen concept employs bacterial superantigens, which are the most potent known activators of T cells, to attract and activate large numbers of T cells to the target [Eisen et al. 2014]. A phase II study of the superantigen-antibody fusion protein targeting 5T4 in RCC showed a significant survival benefit [Shaw et al. 2007], which led to the development of ANYARA (naptumomab estafenatox) [Eisen et al. 2014]. Naptumomab estafenatox has been evaluated in combination with interferon α in a phase II/III clinical trial involving patients with advanced RCC. Although the study failed to meet its primary endpoint, survival benefits were seen in a subgroup of patients with low levels of IL-6 and normal levels of SEA antibodies [Elkord et al. 2015]. The results of this study are still undergoing analysis, and a phase II/III clinical trial of naptumomab estafenatox in combination with a TKI for metastatic RCC is being designed [Stern et al. 2014]. Lastly, promising preclinical activity has been seen with an ADC targeting 5T4 [Sapra et al. 2013], and clinical evaluation is now underway [Stern et al. 2014].

PSA

PSA is a well-known prostate cancer biomarker that is now a target for antigen-specific immunotherapy. An anticancer vaccine known as PROSTVAC^®-VF (PSA-TRICOM; rilimogene galvacirepvec) has been developed that incorporates three co-stimulatory molecules (CD80, CD54 and CD58, collectively referred to as TRICOM) and two recombinant viral vectors encoding PSA transgenes [Singh et al. 2015]. Patients are primed with the vaccinia-based vector and then boosted with a fowlpox vector, both of which are administered with GM-CSF. Early phase I and phase II clinical trials in patients with metastatic castration-resistant prostate cancer (mCRPC) demonstrated that PROSTVAC^®-VF is safe, induces a high rate of immune responses and has survival benefits [DiPaola et al. 2006; Arlen et al. 2007; Gulley et al. 2010]. A double-blind, randomized, controlled phase II clinical trial in patients with mCRPC showed that treatment with PROSTVAC^®-VF significantly improved OS by 8.5 months compared to treatment with control vectors [Kantoff et al. 2010b]. A phase III clinical trial [ClinicalTrials.gov identifier: NCT01322490] in 1200 patients with asymptomatic or minimally symptomatic mCRPC is currently ongoing [Singh et al. 2015].

Survivin

Also known as BIRC5 (baculoviral inhibitor of apoptosis protein repeat-containing 5), survivin is an inhibitor of the intrinsic apoptosis pathway that is widely overexpressed in cancers, making it an attractive target for antigen-specific immunotherapy [Mobahat et al. 2014]. Peptide cancer vaccines targeting survivin have been evaluated in phase I and phase II clinical trials. A phase I study of a multi-epitope anti-survivin peptide vaccine (EMD640744) in patients with advanced solid tumors found an antigen-specific T-cell response in 63% of subjects, and 28% of vaccinated subjects achieved stable disease [Lennerz et al. 2014]. Another phase I study in pediatric brainstem glioma patients using a tripeptide vaccine containing survivin and given in combination with the immunoadjuvant poly-ICLC (polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose) showed evidence of survivin-specific immune responses and clinical benefit [Pollack et al. 2014]. In grade 2 low-grade glioma patients, vaccination with survivin and three other glioma-associated peptides combined with poly-ICLC produced robust T_H1 immune responses [Okada et al. 2015]. The efficacy of another survivin-targeted peptide vaccine in metastatic melanoma patients was evaluated in a phase II clinical trial. Treatment with the vaccine, which contained three survivin peptide epitopes, significantly prolonged OS in patients with antigen-specific T-cell responses [Becker et al. 2012].

IDO1

Upregulation of indolamine-2,3-dioxygenase (IDO1), which catabolizes the essential amino acid tryptophan, is a recently discovered mechanism of tumor cell immune escape. Tryptophan is required for normal T-cell function, and its depletion can lead to immunosuppression [Jiang et al. 2015]. The IDO1 enzyme is overexpressed in various malignancies including breast, NSCLC, prostate and gastric cancers [Uyttenhove et al. 2003]. Two small molecule IDO1 enzyme inhibitors and a peptide-based vaccine are currently being evaluated in clinical trials. Indoximod (1-methly-D-tryptophan) combined with docetaxel was evaluated in a phase I clinical trial in patients with metastatic solid tumors. The treatment was well tolerated with evidence of clinical activity [Soliman et al. 2014]. A phase II trial of this agent in combination with docetaxel as first-line treatment for metastatic breast cancer is ongoing [ClinicalTrials.gov identifier: NCT01792050], in addition to a number of other phase II studies in metastatic melanoma, prostate and pancreatic cancers.

The other small molecule IDO1 inhibitor, INCB024360, is currently being evaluated in combination with ipilimumab in a phase I/II study [ClinicalTrials.gov identifier: NCT02077114 (completed)] in metastatic melanoma [Iversen et al. 2015], a phase I/II study [ClinicalTrials.gov identifier: NCT02318277] of INCB024360 in combination with durvalumab in patients with advanced solid tumors [Ibrahim et al. 2015] and a phase Ib study [ClinicalTrials.gov identifier: NCT02298153] in stage IIIB/IV NSCLC in combination with atezolizumab [Cha et al. 2015]. A peptide vaccine targeting IDO1 was evaluated in a phase I clinical trial in patients with metastatic NSCLC. Median OS was approximately 26 months, and long-lasting disease stabilization was observed in 47% of patients. A currently ongoing phase II study in patients with metastatic melanoma is evaluating the effects of a peptide vaccine containing epitopes to survivin and IDO1 in combination with temozolomide [Iversen et al. 2014].

Discussion

With the recent clinical development and therapeutic successes of immune checkpoint inhibitors targeting the CTLA-4 and PD-1/PD-L1 pathways, such as ipilimumab, pembrolizumab and nivolumab, enthusiasm surrounding anticancer immunotherapy has become intense. However, despite the impressive improvements in clinical outcome seen with these agents, the response rates remain modest, but encouraging. Clearly, we must expand our knowledge about how these agents work and the safest way to administer them in order to improve their efficacy. With respect to antigen-specific immunotherapies, the abundance of tumor antigens that have been identified has allowed for the development of multiple monoclonal antibodies and peptide cancer vaccines targeting these antigens, but the responses to these agents as well remain modest, due in part to mechanisms of tumor evasion and immunosuppression, and the fact that TAA expression profiles are patient-specific. With the availability of checkpoint inhibitors, it is now possible to alter the immunosuppressive tumor microenvironment, which could potentially increase the efficacy of antigen-specific immunotherapies. It is thus becoming quite clear that in order to derive the maximum benefit from these novel immunotherapies, we must learn more about how to best use these agents in combination with chemotherapy and with each other. Numerous clinical trials investigating various combinations of immunotherapies and chemotherapy are already underway.

Another point to consider with the use of cancer vaccines is that while they are not often associated with clinically significant objective responses or improvements in PFS, they have been shown to significantly prolong OS, and thus OS may be the most valid clinical endpoint for assessment of the efficacy of these agents [DeGregorio et al. 2012; Dillman, 2015]. Ultimately, personalized immunotherapy, whereby patients are screened for the expression of tumor antigens and PD-1/PD-L1 so that they can be matched with the appropriate antigen-specific agent, checkpoint inhibitor and chemotherapeutic agent, may be the best means of deriving the maximum therapeutic benefits from these treatments [Kao et al. 2015]. The future of personalized cancer immunotherapy has never been more promising, and the results of ongoing clinical trials are eagerly awaited.