Breast cancer immunotherapy: monoclonal antibodies and peptide-based vaccines.

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Review

Breast cancer immunotherapy: monoclonal antibodies and peptide-based vaccines Expert Rev. Clin. Immunol. 10(7), 927–961 (2014)

Elham Mohit*1, Atieh Hashemi1 and Mojgan Allahyari2 1 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2 Recombinat Protein Production Department, Research and Production Complex, Pasteur Institute of Iran, Tehran, Iran *Author for correspondence: Tel.: +98 218 866 5250 Fax: +98 218 866 5250 [email protected]; [email protected]

Recently, immunotherapy has emerged as a treatment strategy in the adjuvant setting of breast cancer. In this review, monoclonal antibodies in passive and peptide-based vaccines, as one of the most commonly studied in active immunotherapy approaches, are discussed. Trastuzumab, a monoclonal antibody against HER-2/neu, has demonstrated considerable efficacy. However, resistance to trastuzumab has led to development of many targeted therapies which have been examined in clinical trials. Monoclonal antibodies against immune-checkpoint molecules that are dysregulated by tumors as an immune resistance mechanism are also explained in this review. Additionally, monoclonal antibodies with the ability to target breast cancer stem cells that play a role in cancer recurrence are mentioned. Here, clinical trials of HER-2/neu B and T cells, MUC1 and hTERT cancer peptide vaccines are also presented. In addition, various strategies for enhancing vaccine efficacy including combination with monoclonal antibodies and using different delivery systems for peptide/protein-based vaccine are described. KEYWORDS: breast cancer • delivery system • human epidermal growth factor receptor 2/neuregulin • monoclonal antibody • peptide-based cancer vaccine

Breast cancer (BC) is the second leading cause of cancer death in the US women. It has been estimated that half of the new worldwide BC cases and 60% of the BC deaths occur in developing countries [1]. Antigens including carcinoma embryonic antigen, human epidermal growth factor receptor 2/neuregulin (HER-2/neu), mucin1 (MUC1), which is hypoglycosylated in adenocarcinomas, carbohydrate antigens (Tn, TF, sialyl Tn), p53, a tumor suppressor gene mutated in cancers and telomerase reverse transcriptase (TERT) are presented in BC cells and have been studied in humans [2]. Around 10–30% of BCs represent HER-2/ neu overexpression, which are strongly correlated with disease recurrence and worse prognosis [3]. Overexpression of HER-2/neu protein is detected by a semiquantitative scale of immunohistochemistry (IHC) and also FISH, which detects amplification (excess copies) of the HER-2/neu gene, expressed as a ratio of HER-2/neu to chromosome 17 and interpreted as positive if FISH is ‡2.2 [4]. In order to increase the treatment efficacy, some targeted agents aiming at HER-2/neu or

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10.1586/1744666X.2014.916211

VEGF receptor (VEGFR) have been under development [5]. Because of using adjuvant systemic therapy including chemotherapy for most patients, endocrine therapy for those with estrogen receptor-positive disease, and more recently trastuzumab for HER-2/neu-overexpressing BCs, the mortality rate of BC has decreased over the last 30 years [6]. Different strategies have been developed to induce potent immune responses for recognizing and eradicating BCs. Cancer immunotherapy is divided into passive immunotherapy including infusion of tumorspecific monoclonal antibodies (mAbs) and adoptive cell therapy as well as active immunotherapy with cancer vaccines, which stimulates specific immune system [2,7]. Due to short halflife, mAbs therapies require frequent and prolonged duration of administration. In addition, it is impossible to apply this strategy in a prophylactic manner in high-risk patients. To circumvent these problems, active immunotherapy with the ability of stimulating the adaptive immune system and generating T-cell memory responses, which can potentially cause longterm benefit, was applied. However, various

2014 Informa UK Ltd

ISSN 1744-666X

927


Review

Mohit, Hashemi & Allahyari

vaccines led to limited clinical efficacy due to cancer- or treatment-related anergy, human leukocyte antigen (HLA)restriction of vaccine-induced cytotoxic T lymphocytes (CTLs) and finally, inappropriate immunologic targeting [8,9]. Many strategies are applied to enhance vaccine efficacy. The purpose of this review is to provide an overview of clinical trials of mAbs- and peptide-based immunotherapies for BC. In addition, different vaccine delivery systems as a novel approach to improve the efficiency of peptide- and proteinbased vaccines will be discussed. An overview of different strategies discussed in this review about BC immunotherapy is shown in FIGURE 1.

efficient targeting of these cells may represent a novel and promising therapeutic strategy for improving outcome in women with both early- and advanced-stage BC. Different agents including mAbs with the ability to target BCSCs are examined in preclinical models and some of them are currently entering clinical trials [15,16]. In this review, the main features of mAbs-based passive immunotherapy against HER-2/neu, IGF-1R, VEGF and immune-checkpoints molecules will be described. Moreover, novel targeted therapies to overcome trastuzumab resistance will be discussed. We will also briefly mention the development of mAbs and antibody constructs, which selectively target BCSCs populations (FIGURE 1).

mAb-based immunotherapy for BC

mAbs target receptor molecules to disturb signaling pathways and target cancer cells by the immune system [10]. Considerable efficacy was seen in HER-2-positive BC (HPBC) using the mAb targeting HER-2/neu (trastuzumab) [11]. However, primary or acquired trastuzumab resistance usually occurs in HER-2-positive metastatic BC (HPMBC). To overcome trastuzumab resistance, several novel targeted agents are under clinical development. The insulin-like growth factor 1 receptor (IGF-1R) is a transmembrane tyrosine kinase, which is overexpressed in many cancers including breast. So this receptor seems to be a potential therapeutic target in cancer treatment. Currently, the efficacy and tolerability of some novel IGF-1R-targeting mAbs including figitumumab, AVE-1642, R-1507, cixutumumab and dalotuzumab are being evaluated in several clinical trials. Positive associations between HER-2 and VEGF expression levels have been reported by several preclinical and clinical studies in HPBC. Bevacizumab as an anti-VEGF strategy has demonstrated promising activity. The activity of bevacizumab is currently under investigation in several trials as both single agent and in combination with other targeted agents and chemotherapy [11]. As high resistance to any type of therapy may occur in heavily pretreated patients, the study of these agents is increasingly proceeding to earlier stages of the disease in the adjuvant and neoadjuvant (presurgical) setting [12]. Immune-checkpoint blockade is a novel strategy for modulating regulators of immunity. Several immune-checkpoint receptors including CTL antigen 4 (CTLA4), programmed death 1 (PD1) as well as T-cell immunoglobulin and mucin protein 3 (TIM3) or their ligands are upregulated in various types of cancers including BCs [13]. Indeed, preclinical and clinical data established in BC show that the blockade of immunecheckpoints by antibodies can enhance endogenous antitumor immunity with the potential to produce durable clinical responses [14]. BC stem cells (BCSCs) are a distinct subpopulation of tumor cells exhibiting self-renewal and tumor initiation capacity. Due to contribution of BCSCs in intrinsic mechanisms of resistance to conventional chemotherapeutics, novel tumor-targeted drugs and radiation therapy, the 928

HER-2-targeting mAbs HER-2 signaling pathways

HER-2/neu is a transmembrane receptor, which belongs to a family of the epidermal growth factor receptors (EGFR) with tyrosine kinase activity. This family also includes HER-1, HER-3 and HER-4 that are involved in regulating cell growth, survival and differentiation through the activation of signaling cascades, including PI3K/Akt and the rat sarcoma oncogene (Ras)/the serine/threoine kinase (Raf)/ mitogen extracellular kinase/mitogen-activated protein kinase. Downstream signaling cascades are activated via homodimerization and/or heterodimerization of members of this protein family (FIGURE 2). Despite other receptors of this group, HER-2/neu can undergo ligand-independent dimerization, and become constitutively active. Therefore, in HPBC an increase of homodimer formation and cellular signaling can directly occur by the overexpression of HER-2/ neu. In addition to functioning as a homodimer, HER-2/ neu can also interact with other EGFR family proteins. A dominant role has been proposed for HER-3/HER-2 heterodimer in initiation of the PI3K/Akt-dependent signaling cascade, which promotes tumor formation through multiple mechanisms [11,17]. Trastuzumab

Trastuzumab is a humanized mAb directed against the HER-2/ neu extracellular domain (ECD). It was initially approved in 1998 for management of patients with HPMBC. Based on prolonged patient’s survival in several randomized trials, use of trastuzumab is now considered as a standard care for HPBC in both the metastatic and the neoadjuvant/adjuvant settings [17]. Trastuzumab is administrated in patients who overexpress HER-2/neu, defined as IHC 31 or FISH 2.2 [4]. Following binding of the antibody to the ECD of the HER-2/neu receptor, trastuzumab acts as an antitumor reagent through a variety of mechanisms, including activation of antibody-dependent cellular cytotoxicity (ADCC) and also suppression of HER-2/neu homodimer signaling. Moreover, trastuzumab binding may lead to the interference with HER-2-mediated downstream signaling processes such as cell proliferation, angiogenesis and DNA repair [17]. Expert Rev. Clin. Immunol. 10(7), (2014)

Review

Breast cancer immunotherapy

BC immunotherapy Peptide based vaccines (active immunotherapy) CD8+ peotide vaccine

Lapatinib and TKIs

E75 Trastuzumab GP2

T-cell epitope


mAb-based immunotherapy (passive immunotherapy)

PI3K/Akt/mTOR pathway HSP90 inhibitors

Trastuzumab-DM1

+

CD4 peotide vaccine

B-cell epitope

AE37

HER2-targeting mAbs

HER2-MVF

Liposome

Virosome

Pertuzumab

Ertumaxomab

AS15

Figitumumab

Depovax (in DPX-0907)

AVE-1642

Liposome (in Stimuvax) Delivery system

Overcoming trastuzumab resistance

IGF-1Rtargeting mAbs1

R-1507 Cixutumumab

peviPRO™ (PEV6)

Cixutumumab Nanogel

Cholesteryl pullulan (CHP)

VEGF-targeting mAbs

Bevacizumab

Ipilimumab Tremelimumab Immune checkpointstargeting mAb

Nivolumab

CT-011

P245 B6H12.2 BCSCs targeting mAbs

Anti-IL-8 Demcizumab Solitomab

Vantictumab

Figure 1. An overview of breast cancer immunotherapy.

In the neoadjuvant setting, the data from two major randomized trials indicated that addition of trastuzumab to chemotherapy significantly improves pathological complete response (pCR) rates in patients with early- or locally advanced-stage BC [18,19]. According to the results of randomized clinical trials in adjuvant chemotherapy, trastuzumab is generally well tolerated and produces a significant benefit in informahealthcare.com

overall survival (OS) when added to, or administered following a chemotherapy regimen [20–22]. Considering cardiotoxicity with trastuzumab, cardiac risk factors should be screened before treatment with trastuzumab. As the majority of cardiac events associated with trastuzumab are asymptomatic decreases in left ventricular ejection fraction, trastuzumab must be avoided, if baseline left ventricular ejection fraction is less than 50% [23]. 929

Review


Ligands


mAbs Figitumumab Cixutumumab Dalotuzumab AVE-1642 R-1507

mAbs Trastuzumab T-DM1 Pertuzumab Ertumaxomab

mAbs Bevacizumab

Extracellular space

Ligand binding domain IGF1R

HER1-4

HER2 P

VEGFR

P Cytoplasm

Kinase domain TKIs Lapatinib Neratinib Afatinib HSP90 inhibitors Tanespimycin Alvespimycin BIIB021 AUY922

PI3K

SOS

PI3K inhibitor GDC-0941

PI3K/Akt inhibitor BKM-120 Ras

Akt

Raf

mTOR

PI3K/Akt/mTOR inhibitor BEZ-235

mTOR inhibitor Everolimus Temsirolimus Ridaforolimus

MEK HSP90 Nucleus MAPK

Proliferation Cell cycle progression Gene transcription

Figure 2. Downstream signaling of the HER family and several novel targeted agents for HER-2-positive metastatic BC. Downstream cascades are activated via homodimerization and/or heterodimerization of members of this protein family triggered by ligand binding to the extracellular domain (with the exception of HER-2/neu, which is always in an open conformation that allows dimerization). HER kinase domains were phosphorylated and resulted in downstream gene expression required for carcinogenesis. The molecular targets of approved therapies as well as several novel agents which are under development for HPMBC are also depicted in the figure. Akt: Protein kinase B; HER: Human epidermal growth factor receptor; HSP90: Heat shock protein 90; IGF-1R: Insulin-like growth factor 1 receptor; mAbs: Monoclonal antibodies; MEK: Mitogen extracellular kinase; Raf: Serine/threoine kinase; Ras: Rat sarcoma oncogene; TKI: Tyrosine kinase inhibitor; VEGFR: VEGF receptor.

However, based on available data resulted from adjuvant setting with trastuzumab, both asymptomatic and symptomatic side effects seem to be treatable and mostly reversible [11]. Moreover, some clinical trials on adjuvant trastuzumab therapy are conducted to investigate the optimal duration of trastuzumab therapy. According to data from the ongoing HERA study, longer trastuzumab therapy is better than the current standard duration of 1 year. In this study, a sequential approach in patients receiving radiation therapy and adjuvant chemotherapy prior to 1 or 2 years of adjuvant trastuzumab has been tested. On the contrary, an improvement in distant disease-free survival was reported by FinHER trial, in which 9 weeks of adjuvant trastuzumab (given concurrently with chemotherapy) was compared with chemotherapy alone [11]. The ongoing Phase I clinical

930

trials using trastuzumab in combination therapy are demonstrated in TABLE 1. Mechanisms of resistance to trastuzumab

Despite the fact that trastuzumab-based treatment strategy either as a single agent or in combination settings has attractive clinical benefits, trastuzumab resistance is one of the major drawbacks of regimens containing this humanized antibody. In fact, about 70% of HPBC patients may have primary resistance to trastuzumab. Additionally, the majority of those who respond to this treatment tend to develop secondary resistance within 1 or 2 years [5]. Resistance to trastuzumab is not completely understood, but is thought to be mediated by several mechanisms including the loss of or increased HER-2/neu

Expert Rev. Clin. Immunol. 10(7), (2014)


Bevacizumab, trastuzumab

Pegylated liposomal doxorubicin hydrochloride (anthracycline antibiotic), cyclophosphamide (nitrogen mustard alkylating agent), trastuzumab

AMG 479 (anti-IGF-1R mAb), trastuzumab

MM-111 (anti-HER-2/HER-3 bispecific mAb), trastuzumab

Trastuzumab, carboplatin (platinum-based antineoplastic agents), carmustine (alkylating agent), cisplatin (platinum-containing anticancer drug), cyclophosphamide, thiotepa (alkylating agent)

Trastuzumab, vinorelbine ditartrate (antimitotic chemotherapy)

U3-1287 (anti-HER-3 mAb), trastuzumab, paclitaxel (tubulin stabilizer)

AMG 386 (anti-angiopioetin peptibody), paclitaxel, trastuzumab, capecitabine (antimetabolites), lapatinib

Trastuzumab, paclitaxel, recombinant IL-12

LJM716 (anti-HER-3 mAb), trastuzumab

Trastuzumab, alvocidib (CDKs inhibitor)

Trastuzumab, erlotinib hydrochloride (reversible TKI)

Trastuzumab, lonafarnib (farnesyltransferase inhibitor), paclitaxel

NCT00095706

NCT00331552

NCT01479179

NCT01097460

NCT00006123

NCT00014430

NCT01512199

NCT00807859

NCT00028535

NCT01602406

NCT00039455

NCT00033514

NCT00068757

I

I–II

I

I

BC

BC

HPBC, male, recurrent and stage IV BCs

Advanced HPBC or gastric cancer

Male BC, recurrent breast, endometrial, gastric, nonsmall-cell lung, ovarian epithelial and small cell lung cancers

BC, breast neoplasms, locally recurrent and MBCs, solid tumors

I

I

MBC

I–II

BC

BC

I–II

I

HER-2 amplified BC, MBC

I

BC

HPBC, male, recurrent and stage IV BCs

I–II

I–II

BC

Disease condition

I–II

Study phase

National Cancer Institute

Completed

Completed

Completed

Terminated

European Organisation for Research and Treatment of Cancer

Jonsson Comprehensive Cancer Center


Novartis Pharmaceuticals

Amgen

Active, not recruiting

Recruiting

Daiichi Sankyo Inc.

Dartmouth-Hitchcock Medical Center

Beth Israel Deaconess Medical Center

Merrimack Pharmaceuticals

M.D. Anderson Cancer Center

University of Washington

Translational Oncology Research International

Sponsor

Recruiting

Completed

Unknown


Withdrawn

Completed

Unknown

Trial status

[161]

[160]

[159]

[158]

[157]

[156]

[155]

[154]

[153]

[152]

[151]

[150]

[149]

Ref.

Akt: Protein kinase B; BC: Breast cancer; CDK: Cyclin-dependent kinase; HDAC: Histone deacetylases; HER-2: Human epidermal growth factor receptor 2; HER-3: Human epidermal growth factor receptor 3; HPBC: HER-2-positive breast cancer; IGF-1R: Insulin-like growth factor 1 receptor; mAb: Monoclonal antibodies; MBC: Metastatic breast cancer; TKI: Tyrosine kinase inhibitors.

Treatment

Clinicaltrials. gov identifier

Table 1. Ongoing Phase I clinical trials with trastuzumab in patients with HER-2-positive breast cancer.



Review

931

932

MK2206 (Akt inhibitor), trastuzumab, lapatinib ditosylate

Trastuzumab, lapatinib ditosylate

Cetuximab, trastuzumab

Recombinant IL-12, ABI-007 (albumin-stabilized nanoparticle paclitaxel), carboplatin, trastuzumab

Trastuzumab, docetaxel (tubulin stabilizer), gefitinib (EGFR inhibitor)

Vorinostat (HDAC inhibitor), trastuzumab

Docetaxel, lapatinib, trastuzumab

NCT01705340

NCT00085020

NCT00367250

NCT00004074

NCT00086957

NCT00258349

NCT00450892

I

I

I–II

I–II

I–II

I

I

I

I

BC, gastric, bladder, ovarian, non-small-cell lung cancers

Neoplasms, BC

BC

BC, male, recurrent, stage IIIB, IIIC and IV BCs

BC

Inflammatory, male, recurrent, stage IIIA, IIIB and IV BCs

MBC

BC

Adenocarcinoma of the gastroesophageal junction, HPBC, male and recurrent and stage IIIC BCs, stage IIIC and IV esophageal or gastric cancers

BC

Brain and CNS tumors, BC

I–II

I–II

HPBC, inflammatory, male, recurrent and stage IV BCs

Disease condition

I

Study phase

Recruiting



Completed, has results

MacroGenics

GlaxoSmithKline

European Organisation for Research and Treatment of Cancer


City of Hope Medical Center


Completed


Medical University of Vienna

Unknown

Jonsson Comprehensive Cancer Center


Terminated

Completed

New York University School of Medicine

OHSU Knight Cancer Institute


Sponsor

Completed

Withdrawn

Recruiting

Trial status

[173]

[172]

[171]

[170]

[169]

[168]

[167]

[166]

[165]

[164]

[163]

[162]

Ref.

Akt: Protein kinase B; BC: Breast cancer; CDK: Cyclin-dependent kinase; HDAC: Histone deacetylases; HER-2: Human epidermal growth factor receptor 2; HER-3: Human epidermal growth factor receptor 3; HPBC: HER-2-positive breast cancer; IGF-1R: Insulin-like growth factor 1 receptor; mAb: Monoclonal antibodies; MBC: Metastatic breast cancer; TKI: Tyrosine kinase inhibitors.

MGAH22 (anti-HER-2 mAb)

Trastuzumab, pegylated liposomal doxorubicin hydrochloride

NCT00006825

NCT01148849

Trastuzumab, carboplatin, methotrexate (antimetabolite drug), sodium thiosulfate (reducing agent)

NCT00397501

Lapatinib, carboplatin, trastuzumab, paclitaxel

Entinostat (HDAC inhibitor), lapatinib ditosylate, trastuzumab

NCT01434303

NCT00367471

Treatment

Clinicaltrials. gov identifier

Table 1. Ongoing Phase I clinical trials with trastuzumab in patients with HER-2-positive breast cancer (cont.).


Review Mohit, Hashemi & Allahyari




expression, overexpression of HER-1, HER-3 or TGF-a and steric hindrance of HER-2–antibody interaction via expression of mucin 4 glycopeptide. Moreover, cleavage of ECD fragments from the HER-2/neu receptor leads to inhibition of trastuzumab binding. Mutations in the PIK3CA gene and/or loss of PTEN, which suppress the activation of the PI3K/Akt pathway, might lead to constitutive activation of this signaling pathway. Another important source of trastuzumab resistance is activation of the PI3K/Akt pathway via interaction between HER-2/neu and IGF-1R, which is overexpressed in trastuzumab resistance. In addition, in vitro studies showed an increase in the activity of the GTPase p21-rac and Met receptor tyrosine kinase by downregulation of the cyclin-dependent kinase p27kip1, which have been implicated in trastuzumab resistance [24]. Novel targeted therapies to overcome trastuzumab resistance

Many new agents are under clinical development to overcome resistance to trastuzumab therapy. These agents including novel antibody-based approaches against HER-2/neu, newer ErbBfamily tyrosine kinase inhibitors (TKIs), heat shock protein 90 (HSP90) inhibitors, anti-angiogenic therapies, PI3K and mTOR inhibitors and IGF-1R inhibitors are currently being examined in clinical trials in HPBC (FIGURE 2) [24]. TABLE 2 summarizes the ongoing clinical trials of novel therapeutic agents in patients with HPBC. Lapatinib & TKIs

Lapatinib is a small, highly specific, reversible and dual TKIs of EGFR/HER-1 and HER-2/neu, currently approved by the US FDA for use in MBC. TEACH (lapatinib evaluation after chemotherapy) and ALTTO (adjuvant lapatinib and/or trastuzumab treatment optimization), two major adjuvant trials of lapatinib, are now ongoing in patients with advanced-stage disease. The data from these two Phase III trials as well as another Phase II CHERLOB trial (activity evaluation of preoperative taxane– anthracycline chemotherapy in combination with trastuzumab, lapatinib, or combined treatment of trastuzumab and lapatinib) indicated that the combination of trastuzumab and lapatinib demonstrates significantly superior efficacy to either agent used alone. In addition, in the neoadjuvant setting, based on results from several Phase III randomized trials including GEPARQUINTO (evaluation of docetaxel activity in combination with either trastuzumab or lapatinib after epirubicin/cyclophosphamide therapy) and Neo-ALTTO (evaluation of the neoadjuvant therapy of lapatinib, trastuzumab or the combination of both followed by paclitaxel before surgery and the adjuvant chemotherapy followed by the same neoadjuvant therapy after surgery), the combination of trastuzumab and lapatinib remarkably improved pCR rates in patients with early-stage BC [11]. These data suggest that lapatinib may be most optimally used when combined with trastuzumab, with manageable toxic effects. Of note, toxic effects in the lapatinib-treated arm may lead to discontinuation of therapy in some patients. Newer TKIs with the ability to be irreversible and/or active in a broader spectrum are in development for patients with HPBC. A Phase II trial of single-agent neratinib, an informahealthcare.com

Review

irreversible HER-1/HER-2 TKI, was enrolled by both trastuzumab-refractory and trastuzumab-naı¨ve patients and demonstrated a 56% response rate (RR) in trastuzumab-naı¨ve patients and 24% RR in women previously treated with trastuzumab [25]. Currently, neratinib as a single agent or in various combinations (with trastuzumab [26], capecitabine [a prodrug that is enzymatically converted to 5-fluorouracil antimetabolite chemotherapy agent in the body] [27], paclitaxel [a mitotic inhibitor drug] [28], neratinib/paclitaxel vs trastuzumab/paclitaxel [29] and vinorelbine [an antimitotic chemotherapeutic drug] [30]) is being evaluated in HPMBC. Afatinib is another irreversible, smallmolecule ErbB-family TKI, which has demonstrated antitumor efficacy in patients with trastuzumab-refractory HPBC [31]. Recently, HPMBCs were evaluated in a global Phase III trial (LUX-Breast 1) to investigate vinorelbine/afatinib versus vinorelbine/trastuzumab [32]. Sunitinib is a TKI targeted to VEGFR, platelet-derived growth factor receptor and stem cell factor receptor (c-kit). Currently, sunitinib and trastuzumab combinations are being assessed by two early-phase clinical trials in HPBC (sunitinib plus trastuzumab/docetaxel [33] and sunitinib plus trastuzumab [34]) (TABLE 2). PI3K/Akt/mTOR pathway inhibitors

Deregulation of PI3K/Akt/mTOR pathway is thought to be a cause of resistance to HER-2-targeted therapies; so, modulation of this pathway is another strategy to combat trastuzumab resistance. Four main groups including PI3K inhibitors, mTOR inhibitors, dual PI3K–mTOR inhibitors and Akt inhibitors are thought to interfere with the PI3K pathway. Evaluation of everolimus, an mTOR inhibitor, in combination with paclitaxel/trastuzumab or vinorelbine/trastuzumab showed clinical activity in HPMBC [35,36]. Many clinical trials of everolimus in BC patients with low-to-moderate HER-2/neu expression have now been initiated. Ongoing Phase I–II trials are assessing GDC-0941, one of several PI3K inhibitors, in combination with paclitaxel, carboplatin and bevacizumab in HPMBC, as well as along with trastuzumab-DM1 (T-DM1) in patients with trastuzumab-resistant HPBC [11]. Early-phase clinical trials are in progress to evaluate modulation of the PI3K/Akt/mTOR pathway using BEZ-235 (PI3K/Akt/mTOR inhibitor) [37] and BKM120 (PI3K/Akt inhibitor) [38]. Other mTOR inhibitors such as temsirolimus and ridaforolimus are also in clinical development stage in BC patients (TABLE 2) [11]. HSP90 inhibitors

Applying HSP90 is another novel therapeutic approach for circumventing the HER-2/neu resistance in patients with HPBC. HSP90 is a molecular chaperone that regulates the stability and maturation of various oncoproteins including HER-2/neu. Tanespimycin, an HSP90 inhibitor, has demonstrated robust antitumor activity when given in combination with trastuzumab to patients with HPMBC [39]. An overall RR of 22% and clinical benefit rate of 59% were observed in a subsequent Phase II study of this combination [40]. Moreover, there was evidence of alvespimycin antitumor activity, another HSP90 inhibitor, when used 933

934

Study phase

II

II

III

III

III

NeoSphere [NCT00545688]

TRYPHAENA [NCT00976989]

APHINITY [NCT01358877]

PHEREXA [NCT01026142]

MARIANNE [NCT01120184]

Pertuzumab, trastuzumab, docetaxel, paclitaxel, T-DM1

Trastuzumab, pertuzumab, capecitabine

Pertuzumab, trastuzumab, chemotherapy

Pertuzumab, trastuzumab, docetaxel, 5-fluorouracil/epirubicin/ cyclophosphamide, carboplatin, trastuzumab

Pertuzumab, trastuzumab docetaxel

Pertuzumab, trastuzumab, docetaxel

Treatment

II

I

II

[NCT01745965]

[NCT02038010]

[NCT01835236]

Trastuzumab, pertuzumab, paclitaxel, vinorelbine, T-DM1

BYL719 (PI3K inhibitor), T-DM1

T-DM1, trastuzumab

T-DM1, lapatinib, capecitabine

III

ALTTO [NCT00490139]

Lapatinib, trastuzumab

Lapatinib

Ertumaxomab

BC, neoplasms

BC, neoplasms

MBC, advanced BC

HPMBC

HPMBC, recurrent BC, stage IIIA, IIIB, IIIC and IV BCs

BC



Terminated

Recruiting

Not yet recruiting

Recruiting



BC

BC


BC



BC

BC



Trial status

BC

MBC

Disease condition

GlaxoSmithKline

GlaxoSmithKline

Neovii Biotech

Swiss Group for Clinical Cancer Research

Northwestern University

West German Study Group

Hoffmann-La Roche

Hoffmann-La Roche

Hoffmann-La Roche

Hoffmann-La Roche

Hoffmann-La Roche

Hoffmann-La Roche

Genentech

Sponsor

[178]

[177]

[57]

[176]

[175]

[174]

[43]

[56]

[55]

[54]

[53]

[52]

[51]

Ref.

Akt: Protein kinase B; BC: Breast cancer; HPMBC: HER-2-positive metastatic breast cancer; HSP90: Heat shock protein 90; IGF-1R: Insulin-like growth factor 1 receptor; MBC: metastatic breast cancer; TKIs: Tyrosine kinase inhibitors.

III

II

TEACH [NCT00374322]

Lapatinib

TKIs

[NCT00522457]

Ertumaxomab (modified antibody)

III

EMILIA [NCT00829166]

T-DM1 (antibody–toxin conjugate)

III

CLEOPATRA [NCT00567190]

Pertuzumab

HER-targeted antibodies

Trial name/ ClinicalTrials.gov identifier

Table 2. Ongoing clinical trials of several therapeutic agents in patients with HER-2-positive breast cancer.





III

III

GEPARQUINTO [NCT00567554]

NeoALTTO [NCT00553358]

Lapatinib, trastuzumab, paclitaxel

Epirubicin – cyclophosphamide, docetaxel, bevacizumab, paclitaxel, everolimus, trastuzumab, lapatinib

Lapatinib, trastuzumab, paclitaxel, fluorouracil, epidoxorubicin, cyclophosphamide

Treatment

I–II

I–II

II

I–II

[NCT00741260]

[NCT00445458]

[NCT00915018]

[NCT00706030]

II

[NCT00243503]

Sunitinib, trastuzumab

Trastuzumab, sunitinib, docetaxel

Afatinib, trastuzumab, vinorelbine

Neratinib, vinorelbine

Neratinib, paclitaxel, trastuzumab

Neratinib, paclitaxel

Neratinib, capecitabine

Neratinib, trastuzumab

Neratinib

I

[NCT00426530]

Everolimus

Everolimus

BC, metastasis

MBC

BC

BC

BC

BC, advanced malignant solid tumors

BC, advanced BC

Advanced BC, advanced malignant solid tumors BC

BC

Advanced BC

BC, neoplasms

Completed


Completed

Completed





Pfizer

Pfizer

Boehringer Ingelheim

Puma Biotechnology, Inc.








GlaxoSmithKline

German Breast Group

GlaxoSmithKline

Sponsor






BC

BC, neoplasms

Completed

Trial status

BC, neoplasms

Disease condition

[36]

[35]

[34]

[33]

[32]

[30]

[29]

[28]

[27]

[26]

[25]

[181]

[180]

[179]

Ref.


I

[NCT00426556]

Everolimus (mTOR inhibitor)

PI3K/Akt/mTOR pathway inhibitors

I

[NCT00372424]

Sunitinib

LUX-Breast 1 [NCT01125566]

III

I–II

[NCT00398567]

Afatinib

II

[NCT00300781]

Neratinib (irreversible HER-1/HER-2 TKI)

II

Study phase

CHERLOB [NCT00429299]

Lapatinib (cont.)


Table 2. Ongoing clinical trials of several therapeutic agents in patients with HER-2-positive breast cancer (cont.).



Review

935

936

Study phase

II

I

I

[NCT01740336]

[NCT00928330]

[NCT00960960]

Trastuzumab, bevacizumab, GDC-0941, paclitaxel

GDC-0941, trastuzumab-MCC-DM1, trastuzumab

GDC-0941, paclitaxel

GDC-0941, fulvestrant, GDC-0980

Treatment

I

I–II

I

I

II

I

II

I

AVE-1642, fulvestrant

Figitumumab

AUY922

BIIB021, trastuzumab

Alvespimycin, trastuzumab, paclitaxel

Tanespimycin, trastuzumab

BKM120

BEZ-235

BC

BC

BC

BC

BC

BC, HPBC, MBC

MBC

BC, advanced solid tumors, Cowden syndrome

MBC, locally recurrent BC

MBC

BC

BC

Disease condition

Terminated

Withdrawn

Completed

Completed

Completed

Terminated


Completed


Completed

Recruiting

Recruiting

Trial status

Sanofi

Pfizer


Biogen Idec

Bristol-Myers Squibb

Infinity Pharmaceuticals, Inc.



Genentech

Genentech

Genentech

Genentech

Sponsor

[187]

[58]

[42]

[41]

[186]

[40]

[38]

[37]

[185]

[184]

[183]

[182]

Ref.


[NCT00774878]

AVE-1642

[NCT00635245]

Figitumumab

IGF-1R pathway inhibitors

[NCT00526045]

AUY922

[NCT00412412]

BIIB021

[NCT00803556]

Alvespimycin

[NCT00817362]

Tanespimycin

HSP90 inhibitors

[NCT01132664]

BKM120 (PI3K/Akt inhibitor)

[NCT00620594]

BEZ-235 (PI3K/Akt/mTOR inhibitor)

II

[NCT01437566]

GDC-0941 (PI3K inhibitor)







II

[NCT00728949]

I

II

[NCT01220570]

[NCT01605396]

III

III

III

III

II

E2100 [NCT00028990]

AVADO [NCT01200212]

RIBBON-1 [NCT00262067]

RIBBON-2 [NCT00281697]

[NCT00444535]

Lapatinib, bevacizumab

Bevacizumab, standard chemotherapy

Bevacizumab, chemotherapy

Taxane, bevacizumab, capecitabine

Bevacizumab, paclitaxel

Docetaxel, trastuzumab, carboplatin, bevacizumab, 5-fluorouracil, epirubicin, cyclophosphamide

Ridaforolimus, dalotuzumab, exemestane

Ridaforolimus, dalotuzumab

Dalotuzumab

Cixutumumab, tamoxifen, anastrozole, letrozole, exemestane, fulvestrant

Cixutumumab, temsirolimus

R-1507

Treatment

BC, neoplasms

MBC

MBC

BC

BC

BC

BC

BC

BC

MBC

Male BC, recurrent BC, stage IV BC

BC

Disease condition


Completed


Terminated

Completed



Completed

Completed


Suspended

Completed

Trial status

GlaxoSmithKline

Genentech

Genentech

German Breast Group

Eastern Cooperative Oncology Group

National Surgical Adjuvant Breast and Bowel Project

Merck Sharp & Dohme Corp.



ImClone LLC


Hoffmann-La Roche

Sponsor

[75]

[72]

[70,71]

[69]

[68]

[67]

[64]

[63]

[62]

[61]

[60]

[59]

Ref.


III

BETH [NCT00625898]

Bevacizumab

Angiogenesis inhibitors

I

[NCT00759785]

Dalotuzumab

I–II

I

Study phase

[NCT00699491]

Cixutumumab

[NCT00882674]

R-1507





Review

937

Review



in a combination therapy with trastuzumab in patients with HPBC after failure of trastuzumab-containing therapy [11]. Other HSP90 inhibitors, including BIIB021 [41] and AUY922 [42] are currently being evaluated in combination with trastuzumab or as monotherapy or in patients with trastuzumab-resistant HPBC (TABLE 2). Trastuzumab-DM1

T-DM1 is an antibody–drug conjugate consisting of trastuzumab covalently bound to an antimicrotubule agent (emtansine, DM1) via a stable linker. This conjugate allows targeted drug delivery to HER-2-overexpressing cells, thereby minimizing exposure of normal tissue. After T-DM1 internalizing, the DM1 moiety is released in cancer cells resulting in microtubule damage and cell death [11,31,43]. The EMILIA is a randomized, open-label, Phase III trial study evaluating efficacy and safety of T-DM1, as compared with lapatinib plus capecitabine. Patients with HER-2-positive locally advanced or MBC previously treated with trastuzumab and a taxane were enrolled in this trial. The results showed that T-DM1 significantly prolongs OS with less toxicity than lapatinib plus capecitabine among randomly assigned patients [43]. The results of another large global Phase III, a three-arm trial assessing T-DM1 versus T-DM1/pertuzumab versus trastuzumab/taxane, in the first-line setting are yet to be released. T-DM1 appears to be well tolerated as a single agent and has also shown clinical activity in multiple Phase I and II studies in patients with HPBC who had previously used a trastuzumab-based regimen [44]. In three Phase II trials conducted to determine the efficacy of singleagent T-DM1 in HPMBC patients heavily pretreated with trastuzumab, lapatinib and chemotherapy, impressive RRs were reported [45–47]. Preliminary results from a randomized Phase II trial of T-DM1 versus trastuzumab/docetaxel in previously untreated HPBC patients showed higher RR and more favorable toxicity profile for T-DM1 compared with trastuzumab/ docetaxel. The single-arm, Phase Ib/II trial investigated the combination of T-DM1 with pertuzumab in patients with previously untreated and relapsed HPMBC. The preliminary data showed a RR of 57.1% in previously untreated patients and a RR of 34.8% in patients with relapsed disease [48]. Ongoing Phase I and II trials with T-DM1 will clarify the role and the place of this therapeutic conjugate in the treatment of HPMBC patients (TABLE 2). Pertuzumab

Pertuzumab, a humanized mAb, binds to the ECD II of HER-2/neu, which is essential for dimerization. It can efficiently inhibit the formation of HER-2/neu-related homo- and heterodimers and consequently their downstream signal pathway such as PI3K/Akt. Like trastuzumab, pertuzumab also stimulates ADCC as one of its major mechanisms of action [49]. Several studies have implicated the antitumor activity and good tolerance of pertuzumab in metastatic, neoadjuvant and adjuvant settings [31]. As a single agent in patients with trastuzumab-refractory HPBCs, pertuzumab showed only 938

moderate efficacy. However, significant clinical responses are reported when pertuzumab is used concurrently with trastuzumab in many patients with HPBC [11,50]. CLEOPATRA is a Phase III, randomized, double-blind, placebo-controlled study assessed the efficacy and safety of pertuzumab plus trastuzumab/docetaxel (the pertuzumab group), as compared with placebo plus trastuzumab/docetaxel (the placebo group) in patients with HER-2-positive first-line MBC. These patients had not received any previous treatment for their MBC. The results of the primary analysis showed clinically meaningful improvement in OS with the pertuzumab group relative to the placebo group. Although data are still not mature, interim analysis of OS showed a strong trend in favor of trastuzumab plus pertuzumab over trastuzumab alone [51]. NeoSphere is a randomized, Phase II neoadjuvant study of pertuzumab and trastuzumab in patients with operable, locally advanced or inflammatory or early-stage HPBC to evaluate the pCR rate of the combination of docetaxel, trastuzumab and pertuzumab. Compared with the other study arms (trastuzumab plus docetaxel), the docetaxel, trastuzumab and pertuzumab was able to nearly double the complete pathological RR (45.8%) in a neoadjuvant setting prior to surgery [5,52]. Overall, these data suggest that results obtained from the NeoSphere study can be validated in a larger and more definitive CLEOPATRA trial. Since trastuzumab and pertuzumab are structurally very similar, when the two drugs are administered concurrently, an additive toxicity might be anticipated. However, no apparent increase in cardiac dysfunction was seen when pertuzumab was given with trastuzumab. TRYPHAENA is a recently published study, which presented the available randomized Phase II trial evaluating the combination of pertuzumab and trastuzumab given to HPBC patients concurrently with different chemotherapeutic agents. Results demonstrated that the pCR rates across the three arms were quite similar [53]. The efficacy and safety of regimens containing pertuzumab, trastuzumab and chemotherapeutic agents in the adjuvant setting for patients with HER-2-positive early BC or MBC are currently under study in the APHINITY [54] and PHEREXA [55] trials. Results of the MARIANNE [56] study in HER-2-positive first-line MBC are also yet to be released. In this trial, patients are assigned to receive the combination of pertuzumab and T-DM1 (TABLE 2). Ertumaxomab

Ertumaxomab is a trifunctional, hybrid mAb that binds to IgG Fcg receptors (FcgRs) type I/III or CD3 on the surface of immune effector cells, as well as to HER-2/neu of tumor cells. Thus, it conducts formation of a tri-cell complex between T cells, accessory cells and HER-2-expressing cancer cells leading to the death of the tumor cells through phagocytosis. Since ertumaxomab and trastuzumab recognize two different HER-2/neu epitopes, in the presence of high level of trastuzumab, ertumaxomab is able to eliminate HER-2-overexpressing tumor cells. Data from a completed Phase I study of the ertumaxomab demonstrated strong immunological responses in patients with HPBC; also antitumor responses were seen in 5 of 15 patients treated in Expert Rev. Clin. Immunol. 10(7), (2014)


this trial [3]. A Phase II trial [57] is going to evaluate the clinical efficacy and safety of ertumaxomab in patients with HPMBC progressing after trastuzumab treatment (TABLE 2).


IGF-1R-targeting mAbs Figitumumab, AVE-1642, R-1507, cixutumumab & dalotuzumab

The IGF signaling pathway represents a potential therapeutic target in cancer treatment. The IGF-1R is a transmembrane tyrosine kinase, which is overexpressed in many cancers including breast. IGF signaling is active in up to 90% of BCs. This receptor mediates motility, proliferation and apoptosis protection and is activated via binding of the circulating ligands, IGF-1 and IGF-II. There is an increasing evidence of crosstalk between the IGF pathway and other targeted pathways including the ErbB/HER-2 pathway in BC. Thus, signaling through the IGF pathway is a potential mechanism of cancer cell resistance to HER-2-directed agents. Clinical trials of IGF-targeting mAbs in advanced solid malignancies demonstrated that these agents appear to be well-tolerated overall as both single agents and in combination with other targeted agents and chemotherapy [31]. Currently, the efficacy and tolerability of figitumumab, mAb against IGF-1R, are being evaluated in a Phase I clinical trial [58]. AVE-1642 is another mAb against IGF-1R being investigated in an ongoing Phase II clinical trial conducted in hormone-sensitive BC. Moreover, in a Phase I neoadjuvant clinical trial, patients with operable, previously untreated HPBC were randomized to receive the R-1507, a fully humanized anti-IGF-1R mAb, as a single agent [59]. Cixutumumab is another IGF-1R-directed mAb, which is currently administrated in several Phase II trials in BC [60,61]. Moreover, in patients previously treated with trastuzumab and an anthracycline and/or a taxane, cixutumumab is being studied with or without capecitabine/lapatinib [11]. Dalotuzumab is a recombinant humanized mAb antibody-targeted IGF-1R being evaluated in several ongoing Phase I and II clinical trials as both single agent or in combination with other agents [62–64]. In a Phase I trial conducted by Di Cosimo et al., the combination of dalotuzumab and ridaforolimus is tolerable and has promising antitumor activity in 5 of 23 BC patients (TABLE 2) [63,65]. VEGF-targeting mAbs Bevacizumab

The overexpression of VEGF has been observed in both earlyand late-stage BCs and is closely linked to poor clinical outcomes. Bevacizumab is a humanized mAb that blocks angiogenesis via binding to circulating VEGF-A and preventing its binding to the VEGFR2. In the USA, bevacizumab was originally granted ‘accelerated approval’ by the FDA for the firstline treatment of MBC in combination with paclitaxel in 2008. Initial approval based on the results of a randomized Phase III trial demonstrated that paclitaxel plus bevacizumab significantly prolonged progression-free survival and RR versus paclitaxel alone. Several randomized trials have been conducted to evaluate the efficacy and safety of bevacizumab in combination with trastuzumab or chemotherapy in patients with informahealthcare.com

Review

MBCs. Assessing adverse cardiac events associated with bevacizumab, cardiac monitoring is incorporated in all of these trials [11,66]. In the randomized Phase III BETH trial, bevacizumab has been successfully combined with trastuzumab in the first-line of metastatic setting and an objective clinical RR of 48% and clinical benefit rate of 60% were reported [67]. The results of E2100 [68], AVADO [69], RIBBON-1 [70,71] and RIBBON-2 [72] studies evaluating bevacizumab plus chemotherapy versus chemotherapy alone showed an improvement in progression-free survival and objective RR, with no significant difference in OS [73]. Other available randomized trials using bevacizumab in addition to chemotherapy were summarized in a recently published meta-analysis study [74]. Several studies have also been conducted to evaluate the safety and efficacy of bevacizumab as combined with lapatinib in treating HPMBC. The results demonstrated improved efficacy compared with lapatinib or bevacizumab alone in these settings (TABLE 2) [31,75]. Immune-checkpoints-targeting mAb Ipilimumab, tremelimumab, nivolumab & CT-011

Genetic and epigenetic changes found in all cancer cells induce the large number of tumor antigens that the immune system can recognize. In the case of T cells, the response is initiated through antigen recognition by the T-cell receptor and its ultimate duration and amplitude is regulated by a balance between co-stimulatory and inhibitory signals. Immune-inhibitory pathways, termed immune-checkpoints, are normally crucial for maintaining self-tolerance and minimizing collateral tissue damage. Tumors resist immune attack by dysregulation of immune-checkpoints within the tumor microenvironment. Thereby, blockade of immune-checkpoints is one of the most promising approaches to activate therapeutic antitumor immunity [76,77]. CTLA4 and PD1 are two immune-checkpoint receptors that have been most actively studied and have regulated immune responses by different mechanisms [77]. CTLA4, a homolog of CD28, is expressed exclusively on T cells and primarily regulates the amplitude of the early stages of T-cell activation. However, the specific pathways by which CTLA4 dampens T-cell activation are still under investigation. In terms of signaling, inhibitory signal to CTLA4 expressing T cells is transmitted by two major ligands including B7–1 (CD80) and B7–2 (CD86). Studies of CTLA4 knockout mice indicated a significant role for CTLA4 in the development of peripheral tolerance to self-proteins [13,76]. Ipilimumab, an IgG1 antagonistic CTLA4 antibody, was the first FDAapproved immunotherapeutic agent that demonstrated a survival benefit for both untreated and treated refractory metastatic melanoma patients, but is accompanied by frequent immune-related adverse events [78]. Currently, several active clinical trials evaluating ipilimumab in BC patients are under development. The first is being done to evaluate the safety of ipilimumab alone, or given together with cryoablation in women with curable early-stage BC [79]. The other clinical trial of ipilimumab is designed to treat patients with stage IV BC, which has progressed despite treatment with primary 939


Review


therapies [80]. Tremelimumab, a fully human IgG2 antiCTLA4 mAb, enhances human T-cell activation via blocking the CTLA4 binding to CD80 and CD86 [77]. As a single agent, antitumor activity of tremelimumab has been previously reported in patients with advanced melanoma [81]. The first clinical trial of tremelimumab in patients with hormonesensitive MBC has been established in combination with exemestane, an aromatase inhibitor. Combination therapy was tolerable with adverse events including pruritus, diarrhea, constipation and fatigue. Moreover, increased expression of inducible T-cell co-stimulator on T cells also marked increase in the ratio of inducible T-cell co-stimulator-positive T cells to forkhead/winged-helix transcription factor (FoxP3)+ Tregs implicated T-cell activation [82]. PD1 predominantly regulates effector T-cell activity within tissue and tumors via binding to several ligands including PD1 ligand (PDL1) and PD2 ligand (PDL2) expressed by stromal cells, tumor cells or both. Despite some similarities, the synergistic effect of combinatorial blockade of PD1 and CTLA4 implicates non-redundant inhibitory role of these two receptors in maintaining self-tolerance [76,83]. Antibody blockade of PD1 or its ligands has demonstrated enhanced antitumor immunity in mouse models of cancer [84]. A mAb, nivolumab (BMS-936558), specific for PD1, has been reported to have a high RR with durable clinical responses in patients with different advanced-stage cancers including nonsmall-cell lung cancer, renal cell cancer and melanoma. Also it appears to have a much better safety profile than ipilimumab [77,85]. Early-phase clinical trials are in progress to investigate the safety and efficacy of nivolumab as a single agent or in combination with ipilimumab in triple-negative BC (TNBC) and other three tumor types [86]. Other anti-PD1 antibody, CT-011, is currently being evaluated in combination with subcutaneous p53 vaccine in patients with solid tumors that have not responded to standard treatments [87]. Despite conventional chemotherapeutic agents and TKIs, the response to immunecheckpoint inhibitors is slow and in many patients occurs up to 6 months after treatment initiation [77]. TIM3, another immune-checkpoint receptor, inhibits Th1 cell responses via binding to galectin 9, which is upregulated in various types of cancers including BCs [77]. It has also been reported that TIM3 is co-expressed with PD1 on tumor-specific CD8+ T cells and the dual blockade of both molecules significantly enhances antitumor immune responses and tumor rejection in animal models, confirming the notion that combined immunecheckpoint blockade could be a potential treatment strategy to various cancers [88]. Recently, the role of TIM3 was studied in several murine cancer models including 4T1 mammary carcinoma, CT26 colon carcinoma and B16 melanoma [76,88]. Most of the immune-checkpoints inhibitors have shown a dramatic synergy with tumor vaccines in preclinical models. In many poorly immunogenic tumor models, antibodies against both CTLA4 and PD1 strongly enhance the amplitude of vaccine-induced antitumor responses. In combination therapy, cancer vaccines are responsible for generating antitumor T cells, whereas immune-checkpoint inhibitors prevent T-cell anergy [14]. 940

In a preclinical study, mice receiving combination of vaccines and PD1 inhibitor had increased OS and decreased tumor growth [89]. Similarly, in one trial, patients treated with autologous granulocyte-macrophage colony-stimulating factor (GMCSF) secreting tumor cell vaccines were found to have increased inflammatory infiltrates and tumor regression, if they were concurrently treated with CTLA4 inhibitor [90]. However, based on preclinical experiments, multiple additional immune-checkpoints represent promising targets for therapeutic blockade. Moreover, inhibitors for many of these are under active development [77]. BCSCs targeting mAbs P245, B6H12.2, anti-IL-8, demcizumab, solitomab & vantictumab

Cancer stem cells (CSCs) express different cell surface and transmembrane proteins, including CD44, CD47, epithelial cell adhesion molecule (EpCAM, CD326), CD123, CD133, GD2, Lgr5, IGF-IR and members of the Notch and Wnt signaling pathways. These proteins are mainly used for the characterization of CSCs in experimental settings. In recent researches, different agents that can selectively target CSCs have been discovered [15]. Herein, mAbs and antibody constructs directed against BCSCspecific cell surface molecules are discussed. It was reported that P245, mAb raised against human CD44, reduced tumor growth and eliminated BCSCs in xenograft mice with human TNBC. In addition, B6H12.2 (antiCD47) administration prevented and inhibited tumor growth in xenograft mice with patient-derived glioblastoma and ovarian, breast, colon and bladder cancer [15]. Delta-like 4 ligand, an important part of the Notch signaling pathway, plays an important role in stem cell self-renewal and vascular development [91]. It has been demonstrated that demcizumab (OMP-21M18), a humanized mAb against delta-like 4 ligand, inhibits tumor growth, delays tumor recurrence and decreases the frequency of BCSCs in xenograft mice with patient-derived TNBC [15]. It has also been found that antibodies against the IL-8 receptor CXCR1 can target BCSCs in xenograft models inhibiting tumor growth and metastasis [16]. Solitomab (MT110, anti-EpCAM/anti-CD3) is a bispecific T-cell engager designed to link EpCAM expressing cells and T cells causing T-cell activation and a CTL response against EpCAM+ cells. ex vivo treatment of malignant pleural effusions obtained from advanced BC patients with MT110 led to a specific lysis of pleural EpCAM+ BC cells showing the treatment ability of MT110 in BC patients [15,92]. Currently, an openlabel, multicenter dose-escalation Phase I study is ongoing to investigate the safety and tolerability of a continuous infusion of MT110 in locally advanced, recurrent or metastatic solid tumors (including BC), which commonly expresses EpCAM and is not amenable to curative treatment [93]. Vantictumab (OMP-18R5) is a humanized mAb targeting the Wnt pathway by binding to the ECD of Frizzled receptor 1, 2, 5, 7 and 8. It was found that Wnt pathway inhibition via Frizzled receptor targeting results in decreased growth and tumorigenicity of human tumors. Preclinical study indicated Expert Rev. Clin. Immunol. 10(7), (2014)



that OMP-18R5 has the ability to reduce the frequency of CSCs in patient-derived pancreatic and breast carcinomas established in xenograft mice [15,94]. An open-label Phase Ib dose-escalation study is currently recruiting patients with locally recurrent BC or MBC to assess the safety, tolerability and pharmacokinetics of OMP-18R5 when combined with paclitaxel [95]. Finally, the clinical use of these mAbs targeting CSCs or their combination with conventional cytostatic drugs, novel tumor-targeted drugs and radiation therapy may be a beneficial strategy for eradicating CSCs and therefore preventing tumor recurrence and metastasis [15]. Peptide-based vaccines for BC

In order to create a successful tumor vaccine, further to the selection of a proper tumor-associated antigen (TAA) as vaccine target, the complexity of the tumors, the tumor environments and their mechanisms for escaping immunotherapy should be considered. Additionally, the TAAs should be delivered in a manner to increase their immunogenicity. Some adjuvant systems can be added to cancer vaccines to enhance antitumor immune responses and neutralize immunosuppressive activity of the tumor microenvironment [96]. Different cytokines including GM-CSF, IL-2 and type I interferons (IFNs) are combined as immunomodulatory compounds to synthetic peptide vaccines. Herein, GM-CSF for its ability to cause proliferation of dendritic cells and macrophages was co-administrated with peptide-based BC vaccines in clinical trials [97]. HER-2 peptide-based vaccines were divided into B- and Tcell peptide epitopes. T-cell peptide vaccines consist of CD8+ T-cell and CD4+ T-cell epitopes, which bind to MHC class I and MHC class II, respectively. B-cell peptide epitopes are MHC-independent vaccines that preferentially induce antibody responses with antitumor activity against HER-2/neu, as explained for the trastuzumab [96]. Due to different mechanisms of vaccine actions compared with mAbs, vaccines do not necessarily need overexpression of the target protein. Therefore, patients who are either 1–21 for IHC or 0.5%), ERI increased in 85% of patients after boosting. However, ERI did not change after boosting in patients who already had ERI. Reversely, the ELISPOT and in vivo local reaction data indicated an increased response in patients boosted early independent of E75-specific CD8 lymphocyte level. Moreover, the results suggested that 6 months after completion of the primary vaccination series is the optimal time for boosting and may be most effective [101]. The E75 vaccination demonstrates a strong trend toward preventing BC recurrence in vaccinated patients, at the end of the 5-year follow-up period [102]. When the length of follow-up for the trials was extended to 5 years, additional analyses were conducted to evaluate DFS at 24 months and subsequently to provide important information regarding which patients may benefit most from vaccination with E75 peptide + GM-CSF. Furthermore, based on the data obtained in this exploratory Phase I–II clinical trial from 24-month landmark analysis, the appropriate patient population for enrollment of Phase III trial was defined. The obtained data from this analysis showed that patients Expert Rev. Clin. Immunol. 10(7), (2014)


Breast neoplasms, ovarian neoplasms, prostatic neoplasms

I I

540–548 telomerase peptide vaccine + Montanide ISA-51 + GM-CSF

DPX-0907 (7 tumor-specific HLA-A2-restricted peptides + a universal T-helper peptide + a polynucleotide adjuvant + a liposome and Montanide ISA51 VG)

I I and II

dHER-2 (different dose) + of AS15 (fixed dose)

dHER-2 + AS15 ASCI + Lapatinib

MBC

Neoplasms BC

MBC neoplasms, BC

Completed

Completed

NCT00952692

NCT00058526

NCT00140738

NCT01095848

Unknown

Completed

NCT00079157

NCT01660529

NCT00573495

NCT00925548

NCT00004156

NCT00986609

ClinicalTrials.gov identifier

Unknown

Recruiting

Completed

Terminated

Completed


Trial status

Michael Morse

GlaxoSmithKline

GlaxoSmithKline

ImmunoVaccine Technologies, Inc.

University of Pennsylvania

Abramson Cancer Center of the University of Pennsylvania

Abramson Cancer Center of the University of Pennsylvania

EMD Serono

Memorial Sloan-Kettering Cancer Center

Joseph Baar

Sponsor

BC: Breast cancer; GI: Gastrointestinal; hTERT: Human Telomerase reverse transcriptase; HLA: Human leukocyte antigen; KLH: Keyhole limpet hemocyanin; MBC: Metastatic breast cancer; MUC1: Mucin1; GM-CSF: Granulocyte-macrophage colony-stimulating factor; poly-ICLC: Polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose.

I

dHER-2

dHER-2 (HER-2/neu protein)

BC (stage IV)

–

hTERT/Survivin multi-peptide vaccine + basilixumab + Prevnar

MBC

I

Breast neoplasm, BC, carcinoma, ductal (advanced BC)

Endocrine-sensitive advanced BC

BC

BC, inflammatory BC, stage I, II, IIIA, IIIB, IIIC B. C, TNBC

Disease condition

hTERT/Survivin multi-peptide vaccine

hTERT

III

I

MUC1 + KLH + QS-21

Stimuvax (L-BLP 25 or BLP25 liposome vaccine) + hormonal treatment

0

Study phase

MUC1 + poly-ICLC

MUC1

Treatment

Table 5. Examples of clinical trials using mucin1 and human telomerase reverse transcriptase peptide as well as dHER-2 protein.


[10]

[203]

[202]

[133]

[201]

[130]

[128]

[123]

[119]

[121]

Ref.


Review

945


Review


with low HER-2/neu expression tumors benefited from vaccination and indicated statistically significant improvement in DFS rates compared with controls. Therefore, the Phase III trial will enroll lymph node-positive patients, as they are at higher risk for disease recurrence. In addition, only patients with HER-2/neu low-expressing tumors, defined as IHC 1+ or 2+ or FISH 1.4, will be appropriate for Phase III trial enrollment, as these patients have the most robust response to vaccination. The eligible patients will be randomized to two arms: E75 peptide + GM-CSF or GM-CSF alone [8]. Currently, to assess the efficacy and safety of E75 peptide + GM-CSF for preventing BC recurrence and also to evaluate and compare the DFS in the vaccinated and control subjects, a multicenter, multinational, prospective, randomized, doubleblind, controlled Phase III clinical trial [103] is recruiting (TABLE 4) [98]. GP2

GP2, a 9 amino acids peptide derived from the transmembrane portion of the HER-2/neu protein, demonstrated low binding affinity to the HLA-A2 molecule, in contrast to E75. This peptide is considered as a subdominant epitope of HER-2/neu. However, GP2 can induce a CTL response similar to E75, recommending that GP2 is equally or perhaps more immunogenic. It is assumed that GP2 peptide can stimulate a different and perhaps more varied population of CD8+ T cells. If so, then GP2 vaccination may prove to be more effective than E75 vaccination [104]. The first Phase I clinical trial of GP2 vaccine was conducted in disease-free NN BC HLA-A2-positive patients. GP2-specific CD8+ T cells were elicited after GP2 vaccination in all patients. In addition, GP2 vaccination also caused epitope spreading against E75, which was proved by enhancement in E75-specific CD8+ T cells [104]. To investigate the effect of GP2 vaccine on prevention of BC recurrence, clinically diseasefree NP or high-risk NN patients with any level of HER-2/neu expression were enrolled in a randomized, multicenter Phase II clinical trial of GP2 vaccine [105]. In this study, patients were randomized to one to four treatment arms, HLA-A2-positive patients received: GP2 peptide + GM-CSF, or GM-CSF alone and HLA-A2-negative patients received; AE37 + GM-CSF, or solely GM-CSF. The minimal toxicity was comparable between the GP2 peptide + GM-CSF and GM-CSF alone, recommending the critical role of GM-CSF. In addition, a >50% reduction in BC recurrences in patients receiving GP2 peptide + GM-CSF has correlated with increased in vivo immune response [106]. CD4+ peptide epitope AE37

Production of CD8+ CTL response by MHC class I peptides is relatively short-lived and induces tolerance due to lack of costimulatory signals most likely via anergy [100]. Strong responses from CD4+ T-helper (Th) cells enhance CD8+ responses and induce long-term immunologic memory and persistently stimulate CTLs [107]. MHC class II peptides are more promiscuous 946

and may be more generally applied. There may be no necessity for HLA-typing in patients before administration [100]. The weak presentation of MHC class II epitope peptides is a major matter of cancer peptide vaccines in clinical trials. In order to enhance peptide presentation to CD4+ T cells, the immunoregulatory segment of the MHC class II-associated invariant chain (Ii) was coupled to MHC class II epitope. Specifically, the covalent addition of the shortest active Ii-Key peptide sequence, LRMK, to MHC class II epitopes strongly facilitates direct antigenic epitope charging of major MHC class II molecules at the cell surface and thus enhances in vitro and in vivo presentation of the tethered peptide [108]. AE37 is the Ii-Key hybrid of HER-2/neu peptide 776–790 (AE36) (FIGURE 3). Sotiriadou et al. demonstrated that AE37 induced more effective immunological responses over the corresponding native peptide in T-cell cultures from patients with various types of HER-2+ tumors [107]. The results of the first Phase I clinical trial of this hybrid vaccine in disease-free, NN BC patients indicated that the AE37 vaccine seems to be safe and elicits a HER-2-specific immune response, even without using an immunoadjuvant. Additionally, a Phase II study [105] is ongoing to determine if the AE37 peptide + GM-CSF vaccine reduces the recurrence rate in HLA-A2-negative, HER-2/neu-positive, NP or high-risk NN BC patients. Due to significant local/systemic toxicity, decreasing GMCSF dose for subsequent inoculations defined as dose reductions (DRs) was made in clinical trials using different immunogenic HER-2/neu peptides (AE37/E75/GP2) + GM-CSF. DR patients showed greater immune responses both ex vivo and in vivo. Therefore, optimal strategy for vaccine dosing should be applied in peptide-based cancer vaccines [109]. The optimal biologic dose was determined to be 500 mg of peptide with GM-CSF >30 mg and

Monoclonal antibodies and tumor vaccines.

Current modalities in cancer immunotherapy: Immunomodulatory antibodies, CARs and vaccines.

Biological agents in cancer therapy: cytokines, monoclonal antibodies and vaccines.

HER2 and immunotherapy using monoclonal antibodies in colorectal cancer.

Anti-idiotype monoclonal antibodies as vaccines for human cancer.

Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy.

Potential of genetically engineered monoclonal antibodies for cancer immunotherapy.

Production of monoclonal antibodies in plants for cancer immunotherapy.

Cancer immunotherapy. Neo approaches to cancer vaccines.

Immunotherapy of cancer: from monoclonal to oligoclonal cocktails of anti-cancer antibodies: IUPHAR Review 18.

RNA-Based Vaccines in Cancer Immunotherapy.

Bispecific antibodies in cancer immunotherapy.

Immunomodulatory Monoclonal Antibodies in Combined Immunotherapy Trials for Cutaneous Melanoma.

Future prospects of monoclonal antibodies as magic bullets in immunotherapy.

Vaccines and early breast cancer.

Breast Cancer Immunotherapy.

Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunotherapy.

Two monoclonal antibodies identify antigens preferentially expressed on normal human breast cells versus breast cancer cells.

Multi-specific antibodies for cancer immunotherapy.

The prognostic value of immunoperoxidase staining with monoclonal antibodies NCRC-11 and 3E1.2 in breast cancer.

Treatment of gastrointestinal cancer using monoclonal antibodies.

Immune modulation by chemotherapy or immunotherapy to enhance cancer vaccines.

Monoclonal antibodies against human cancer stem cells.

Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus.