Current Cancer Drug Targets, 2011, 11, 85-102 85

1568-0096/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.

Cancer Vaccines in Phase II/III Clinical Trials: State of the Art and

Future Perspectives

S. Cecco#,1

, E. Muraro#,2

, E. Giacomin1, D. Martorelli

2, R. Lazzarini

1, P. Baldo

*,3 and

R. Dolcetti2

1Pharmacy Unit;

2Cancer Bio-Immunotherapy Unit;

3Drug Information Centre National Cancer Institute CRO-IRCCS,

Aviano, Italy

Abstract: The topic of this review covers a very important branch of cancer research, cancer vaccination. The growing

knowledge in tumor immunology has evolved rapidly, starting from unspecific generic stimulation of the immune system

to more specific approaches based on the availability of tumor antigens. The review covers molecular and cell biology,

and pharmaceutical technology of cancer vaccines. Particularly, it is aimed at highlighting the results of cancer vaccines

from phase II and III clinical trials, an issue that is of relevance to better understand how cancer vaccines can successfully

complement antitumor therapy, including conventional chemotherapy and the recently developed target-based drugs.

Keywords: Cancer, therapeutic vaccines, immunotherapy, clinical trials, investigational drugs, tumor-associated antigen.

INTRODUCTION

Cancer is a major cause of morbidity and mortality worldwide and, importantly, the WHO indicates that at least 30–40% of all cancer deaths will be preventable in the next years [1]. Traditional modalities for the treatment of patients with cancer included surgery, chemotherapy and radiation therapy, but immunotherapy has emerged in the last decades as an innovative and alternative tool in the fight against ma-lignancies [2].

The immune system is an organization of molecules and cells with specialized roles in defending against foreign agents. There are two fundamentally different types of im-mune responses to invading non-self antigens (ags): a) innate (or natural) responses that are generally activated at compa-rable levels each time the exogenous agent is encountered, whereas b) acquired (or adaptive) responses improve on repeated exposure to a given antigen. The innate responses use phagocytic cells (neutrophils, monocytes, and macro-phages), cells able to release inflammatory mediators (baso-phils, mast cells, and eosinophils), and natural killer (NK) cells. The molecular components of innate responses include complement, acute-phase proteins, and cytokines such as the interferons (IFNs). Acquired responses involve the prolifera-tion of ag-specific B and T cells, which occurs when the sur-face receptors of these cells bind to the cognate ag. Special-ized antigen-presenting cells (APCs) display the ag to lym-phocytes on human leukocyte antigen (HLA) molecules and cooperate with them in the response to the ag. B cells re-spond to the immunogens by secreting immunoglobulins, the ag-specific antibodies mainly responsible for the elimination of extracellular agents.

Although T cells can help the B counterpart to make an-tibodies, they also contribute to the eradication of intra-

*Address correspondence to this author at the Hospital Pharmacy, Centro di

Riferimento Oncologico of Aviano, Via F. Gallini, 2, 33081 Aviano (PN),

Italy; Tel: +39 0434 659221; Fax: +39 0434 659743; E-mail: pbaldo@cro.it #Equally contributed.

cellular pathogens by activating macrophages and directly killing infected cells, presenting non-self ags on HLA class I (CD8+ T cells) and HLA class II (CD4+ T cells) molecules. Since cancer is the result of multiple genetic alterations ac-cumulated over many years and the cumulative effect of ge-netic alterations could result in protein structure changes, mutated or differentially expressed proteins represent poten-tial tumor-specific (TSAs) or tumor-associated ags (TAAs), being thus suitable to be targeted by T-cells. Therefore, the expression of TAAs on tumor cells constitutes a fundamental pre-requisite for cancer immunotherapy and a therapeutically relevant opportunity to improve the clinical control of these diseases. Immunotherapeutic strategies include active and passive approaches. Active immunotherapy mainly aims to elicit the body’s own response to attack tumor cells; whereas passive immunotherapy relies on therapeutics that can di-rectly mediate the killing of the tumor (i.e. targeting antibod-ies and adoptive transfer of tumor-specific T lymphocytes are components of passive immunotherapy, whereas cytoki-nes and cancer vaccines are active immunotherapeutic agents).

PROPHYLACTIC AND THERAPEUTIC VACCINES

Vaccination is one of the most successful public health initiatives ever achieved [3]. The use of vaccines for the treatment of cancer is based on the finding that tumors in humans and in animal models express target antigens that can be recognized by immune cells. Unlike vaccination against infectious agents where vaccines are used to induce neutralizing antibodies that act prophylactically, the main goal of therapeutic cancer vaccination is to induce and ex-pand host immune responses able to induce the elimination of established tumors. However, vaccines for most tumor types remained elusive until recently and thus several strate-gies have been devised to deliver specific and immunogenic vaccine components to the immune system in the hope of eliciting a prophylactic or therapeutic immune response. In the last decade, in fact, the molecular identification of TAA from human malignancies and the discovery that tumors can be recognized by the immune system of cancer patients

86 Current Cancer Drug Targets, 2011, Vol. 11, No. 1 Cecco et al.

stimulated an impressive array of immunological approaches for the successful generation of cancer vaccines [4]. These include T-cell epitope peptides, plasmid DNA and recombi-nant viral vector vaccines, allogeneic or autologous whole tumor cell vaccines, dendritic cell (DC)-based vaccines or oncolysates complex vaccines.

Prophylactic (or Preventive) Vaccines

Prophylactic (or preventive) vaccines are intended to prevent cancer developing in healthy subjects. Available preventive cancer vaccines target infectious agents that are causally involved in the development of distinct types of cancer [4, 5] and are expected to behave as traditional vac-cines, which help prevent infectious diseases such as measles or polio. Both cancer preventive vaccines and traditional vaccines are based on the use of ags carried by the infectious agents that are relatively easy for the immune system to rec-ognize as foreign. Fundamentally, two types of cancer pre-ventive vaccines have been successfully developed to date. The first Food Drug Administration (FDA) approved preven-tive cancer vaccine protects against hepatitis B virus (HBV) infection. Chronic HBV infection is the most common viral cause of liver disease worldwide, with over 350 million in-fected people. Hepatocellular carcinoma (HCC) is the main consequence of chronic HBV infection, although a variety of viral and host factors also contribute to its development. Moreover, HCC is one of the most common malignancies in humans and occurs mainly in adults between 40 and 60 years of age with a hepatitis B surface antigen (HBsAg) seroposi-tive rate of 70% to 80% [6]. Available evidence indicates that the development of HCC is favored by increased hepa-tocyte turnover incited by chronic liver injury and regenera-tion. The most effective measure of prevention of HBV-related HCC is the prevention of HBV infection by vaccina-tion and the first HBV vaccine was approved by FDA in 1981, making it the oldest cancer preventive vaccine to be successfully developed and marketed. The first licensed HBV vaccines were plasma-derived and composed of puri-fied HBsAg; the majority of currently used HBV vaccines are produced by recombinant DNA technology [7, 8]. The universal HBV vaccination program activated in Taiwan was associated with a significant reduction in the rates of child-hood HCC subsequent to vaccine introduction. Indeed, through the reduction of HBV sero-prevalence in the general population, the incidence of HCC is likely to decline signifi-cantly, especially in areas where HBV is endemic. The most important preventive strategy’s adoption of the universal HBV vaccination program is now in its third decade and there is a clear reduction in both chronic HBV infection (HBsAg “carriage”) and in childhood HCC, particularly in under-developed world populations [9].

In 2006, the U.S. FDA approved the vaccine known as Gardasil

®, which protects against infection by two types of

oncogenic human papillomaviruses (HPV), types 16 and 18, which are responsible for approximately 70% of all cases of cervical cancer worldwide [10, 11]. Moreover, in 2008, the FDA expanded Gardasil

®’s approval to include its use in the

prevention of HPV-associated vulvar and vaginal cancers. The vaccine’s composition is based on HPV antigens derived from the combination of four different types of virus-like

particles (VLPs), which correspond to HPV types 6, 11, 16, and 18. Unlike traditional vaccines, which are often com-posed of attenuated, whole microbes, these VLPs are not infectious; nevertheless, these particles are still able to stimulate the production of antibodies against all pathogenic HPV types. A second manufactured HPV vaccine named Cervarix

® has also been developed. Unlike Gardasil

®, this is

a bivalent vaccine, composed of HPV types 16 and 18 VLPs, being thus able to provide protection only against these two major HPV types. The public health benefits of vaccines against HPV types 16 and 18 may extend beyond the reduc-tion of the risks of cervical, vaginal and vulvar cancers. In fact, available evidence suggests that chronic infection by one or both of these virus types is also associated with can-cers of the anus, penis, and oropharynx [12]. The results of prospective studies will elucidate the possible protective role of these vaccines also against these additional HPV-associated tumors.

Therapeutic Vaccines

Although vaccines have been quite successful in prevent-ing infectious disease, therapeutic immunization in the set-ting of established, chronic disease, including both chronic infections and cancer, has been much less effective. In con-trast to vaccines against pathogens where the target antigens are foreign to the immune system, the majority of the target antigens of cancer vaccines is close to self antigens and therefore is generally less immunogenic. Therapeutic cancer vaccines thus constitute a unique and highly challenging approach to cancer therapy. These reagents are expected to exert antitumor effects by engaging the host immune re-sponse, and have great potential for circumventing the intrin-sic drug resistance that limits standard cancer management. Distinct vaccine platforms incorporate tumor antigens in different ways to activate tumor immunity. Vaccines can be highly targeted, such as peptide-based vaccines, or less well-defined, such as whole tumor cells or tumor cell lysates, but vaccines platforms are mainly designed to specifically ma-nipulate B cells, T cells, or professional APCs [13]. The emerging and most promising molecular and cellular plat-forms will be briefly discussed below and shown in Fig. (1).

MOLECULAR BIOLOGY CANCER VACCINES (CV) PLATFORMS

Protein- or Peptide-Based Vaccines

Protein- and peptide-based vaccine strategies are based on the administration of high doses of the antigenic pep-tide(s) in order to successfully load empty major histocom-patibility complex (MHC) molecules on APCs in vitro and in vivo. Although the simplest cancer vaccine formulations consist of peptide delivered intra-dermally together with an immunologic adjuvant [3], this vaccine strategy requires knowledge of relevant tumor antigens and the precise defini-tion of MHC class I and MHC class II epitopes. This vaccine platform has a number of advantages over other types of cancer vaccines. Manufacturing proteins or peptides on a large scale is easy and with affordable costs, peptide vac-cines are safe, with no potential for re-assortment, infection, or recombination. On the other hand, drawbacks include the

Cancer Vaccines in Phase II/III Clinical Trials Current Cancer Drug Targets, 2011, Vol. 11, No. 1 87

weak immunogenicity of short peptides, and the strict re-quirement of the appropriate HLA genotype by patients to be vaccinated. The immunogenicity can be however improved by introducing lipid, carbohydrate, or phosphate groups, by introducing protease-resistant peptide bonds to regulate their processing and prolong their half-life in vivo, or by combin-ing the peptides with several adjuvants as ligands of toll-like receptors (TLR), recombinant cytokines, oil-emulsion or polysaccharide adjuvants. Although a number of these vac-cines are under active investigation for the management of infectious disease, cancer, autoimmune disease, allergy, and Alzheimer’s, none has yet been approved for commercial use.

Vaccination with Xenoantigens

The demonstration that a single T-cell receptor (TCR) can efficiently recognize a relatively broad spectrum of re-lated ligands raised the possibility that a T cell recognizing a xenoantigen may cross-react with its self-homologous coun-terpart (i.e. Hb(64-76)/I-Ek) [14, 15]. Considering the TAA, most of the autologous T cells specific to TAAs may have been deleted from the T cell repertoire, but T cells specific for the xenoantigenic counterparts of TAAs may survive the negative selection. These xenoantigen-specific T cells, once induced and activated by immunization with a xenoantigen, may cross-react with their cognate TAA owing to the plastic-

ity of TCR recognition. Therefore, immunization with xenoantigens constitutes a plausible approach to overcome the immune tolerance against homologous self-antigens. Available evidence demonstrates that xenoantigenic immu-nization is effective in the induction of both humoral and cellular immune responses against their self counterpart.

Genetically Engineered Plasmid DNA

DNA vaccines are simple rings of DNA containing a gene encoding for an ag and a viral promoter/terminator to make the gene expressed in mammalian cells. This may be a highly promising approach for generating all types of desired immunity, including cytolytic T lymphocytes (CTL), T helper cells, and antibodies. This approach takes advantage of the well conserved and elegant ability evolved by several viruses to introduce their genetic material directly into target mammalian cells when injected in vivo, with ensuing synthe-sis of the encoded protein repertoire [16]. These DNA plas-mids require no specific formulation or alteration other than the presence of a promoter active in mammalian rather than bacterial cells. Moreover, unlike peptide vaccines, the rele-vant epitopes do not need to be defined, nor is the vaccine limited to particular HLA genotypes, as the whole protein will be processed in vivo by host APC. This type of vaccine is inherently immunogenic due to the presence of immune-stimulating CpG oligodeoxinucleotide (ODN) motifs that

Fig. (1). Molecular and cell biology-based cancer vaccine platforms. Examples of vaccines are reported according to reviewed clinical trials.

For the specific mechanisms of action, refer to clinical trials section.

88 Current Cancer Drug Targets, 2011, Vol. 11, No. 1 Cecco et al.

can bind and activate specific TLRs. As gene delivery vehi-cles, DNA plasmids have a number of advantages over other systems which involve either removal of cells from an indi-vidual in order to transfect them ex vivo prior to re-implantation, or which require the manipulation of viruses and bacteria (which are themselves pathogenic, immuno-genic or both) – a process markedly more complicated than manipulating and producing plasmids. On these grounds, a variety of approaches are under evaluation to increase the potency of DNA vaccines, including strategies able to in-crease the efficiency of transfection or to target the DNA to specific sites, whilst also providing a means to avoid the traditional syringe (to facilitate global administration).

Vectors, Viruses or Bacteria

Since the primary goal of CV strategies is the boosting of antitumor immune responses, the ideal antigen delivery vec-tor should directly infect APCs in vivo and facilitate antigen delivery to both MHC class I and II antigen-presentation pathways. On these grounds, recombinant viral vectors and bacteria represent promising strategies to deliver defined TAA using gene transfer. These approaches include replica-tion-defective pox viruses, adenoviruses, adeno-associated viruses, herpesviruses, retro- or lentiviral vectors, and differ-ent bacteria, as Listeria monocytogenes, Salmonella typhi-murium, Shigella, and Mycobacterium bovis BCG (Bacillus Calmette-Guerin). Despite the high immunogenic potential of these platforms, their toxicity still remains a major disad-vantage, being related in particular to the risk of a productive infection or to unwanted recombination with other infectious agents. It is worth mentioning in this respect that Wu and co-workers recently demonstrated the enhanced potency of vac-cinia vectors carrying the HPV gene E7 fused with the LAMP1 gene, which targets E7 to the MHC class II antigen-processing pathway for presentation to CD4

+ T cells [17,

18]. Interestingly, an HPV-specific CTL response was ob-served in one of the patients included in the study, but unfor-tunately all the patients mounted an anti-vaccinia antibody response [19].

CELL BIOLOGY CANCER VACCINES (CV) PLAT-FORMS

Dendritic Cells “In Vitro” or “In Vivo”

Activated and mature DCs express high levels of MHC class I and II molecules for priming CD8

+ and CD4

+ T cells,

respectively, and additionally supply potent co-stimulatory signals for T-cell activation. By providing a source of tumor ags for these cells, it should therefore be possible to induce a potent tumor-specific immune response using TAA-loaded DCs. On this basis, several approaches using DCs have been tested in animal models, including DC loaded in vitro with peptides [20, 21] or proteins [22], DC fused with whole tu-mor cells and ex vivo transduced DC [23-26]. The in vitro generation of DCs suitable for immunotherapeutic ap-proaches is largely dependent on the stimulation factors that induce their maturation. For example, a particularly effective protocol was recently developed to generate fully mature DCs using an optimized maturation cocktail including dif-ferent growth factors and interleukins [27]. Compared with

standard DCs maturation protocols [28], the cocktail-induced DCs secreted more IL-12p70, also inducing higher numbers of functional CD8

+ T cells against TAA [29], and recruiting

activated NK cells [30]. Taken together, these findings indi-cate the possibility to use cocktail-matured DCs for the de-velopment a new generation of ex vivo-manipulated DCs for future clinical trials.

On the other hand, targeting of ags to DCs in vivo repre-sents a promising approach for DC-based vaccination, as it can bypass the laborious and expensive ex vivo ag-loading and cell culturing, thus facilitating the large-scale application of DCs-based immunotherapy [31]. More importantly, in vivo DCs-targeted vaccination was reported to be more effi-cient in eliciting an anti-tumor immune response and in con-trolling tumor growth in animal models [32, 33]. Encourag-ing results for in vivo-targeted DCs vaccination came from the use of engineered lentiviral vectors encoding the human melanoma antigen NY-ESO-1 [34, 35], thus indicating that targeting ag expression to DCs with lentiviral vectors can provide a safe and effective vaccine.

Autologous or Allogeneic whole Tumor Cells or Lysate-

derivate Vaccines

Vaccines derived from whole tumor cells or from tumor lysates have the advantage to deliver a diverse panel of tu-mor antigens and to simultaneously provide both CD8

+ and

CD4+ T-cell epitopes. These vaccines can be composed of

autologous or allogeneic tumor cells. Autologous tumor cell-derived vaccines potentially include unique TAAs, which should be ideal targets for immunotherapy. Although these approaches are attractive, since they can deliver ags that may be unique to an individual’s tumor and can also directly prime the antitumor immune response, they suffer from the difficulty to obtain tumor cells in sufficient quantities. Therefore, allogeneic tumor cells represent a valid alterna-tive that may circumvent these practical limitations. Alloge-neic tumor cells can be used as a source of TAA, not only because tumors have overlapping antigen expression profiles (or shared antigens) [36, 37], but also because the tumor ag-specific immune response can be initiated by cross-priming [38], bypassing the need to match the MHC haplotype of the patient to the vaccine platform. Moreover, considering the low immunogenicity of tumor cells, they must be modified in a fashion that enhances their immunogenic properties [39], and generally, this can be accomplished by genetic, viral, or chemical modification, or by the co-delivery of a strong adjuvant.

ENHANCING THE IMMUNOGENIC POTENTIAL OF CVS: THE ROLE OF ADJUVANTS

Most of the described CVs have the main disadvantage to target self-ags being thus poorly immunogenic. To overcome this limitation, CVs have often been associated with sub-stances able to enhance their immunogenic potential, named adjuvants. Adjuvants (adiuvare is Latin for “to help”) are broadly defined as pharmacological or immunological agents able to modify the effect of other agents (e.g. drugs, vac-cines), having few or none direct effect when given by them-selves. In immunology, and in particular in the field of CVs,

Cancer Vaccines in Phase II/III Clinical Trials Current Cancer Drug Targets, 2011, Vol. 11, No. 1 89

adjuvants may exert their enhancing effects according to some fundamental immune-functional activities [40], includ-ing in particular ag localization, its permanence in lymph nodes and APC activation. Some substances are able to fa-cilitate ag transport, uptake and presentation by ag-capturing and -processing cells in the lymph nodes draining the vac-cine injection site. It is the case of lipid matrix-based vac-cines, including liposomes [41], micelles, polymer micro-spheres [42], and immunostimulating complexes (ISCOMs and ISCOMATRIX) that are composed by saponin, choles-terol and phospholipid in a structure of approximately 40nm in diameter, virtually able to deliver any ag [43]. The persis-tence of the ag in the lymph node or at the injection site is the second step necessary to obtain a durable immune re-sponse. This so-called depot effect is supported by oil-based adjuvants [44], gels, polymer microspheres [42] and non-ionic block co-polymers. Other types of adjuvants are able to mimic the conserved microbial structures named pathogen-associated microbial patterns (PAMPs) activating cells from the innate immune system (effect known as Signal 0). This is a likely major mechanism for adjuvants based on microbial components, like pertussin and cholera toxin, recombinant BCG used as delivery vector for heterologous ags [45], mo-nophosphoryl lipid (MPL), a component of endotoxin (lipopolysaccharide) [46], yeast estracts, and CpG ODN [46, 47]. Biological products derived from non-microbial organ-isms can be used as adjuvants too: for example keyhole lim-pet hemocyanin (KLH), a large protein produced by sea animals, is able to increase ags ability to stimulate immune response [48]. Squalene, originally obtained from shark liver oil, is now frequently used as adjuvant in the formulation of flu pandemic vaccines, and has been proposed for CVs be-cause of its ability to stimulate immune response through production of CD4

+ memory cells [49]. Similarly, there are

molecules able to induce tissue destruction or stress, evoking expression of co-stimulatory molecules on the APCs; it seems, in fact, that immune responses are proportionally related to tissue damage. This danger signal is induced by oil-emulsion surface active agents, aluminium hydroxide (AL(OH)3) that is able to induce also a good antibody pro-duction (Th2 response) [50], heat shock proteins (HSP) [51], and Freund’s complete adjuvant (FCA). The latter, com-posed of mineral oil and inactivated mycobacterium, despite the high efficacy demonstrated in animal models, has yet to be approved for clinical use due to its adverse inflammatory effects [52]. Finally, it has been suggested that recombinant analogues of a number of cytokines may exhibit adjuvant activity, recruiting B- and T-cells but also enhancing tran-scriptional events leading to a clear potentiation of the whole immune system. Some examples in this field include type I IFNs that may act directly on B- and CD4

+ T-cells, protect-

ing them from apoptosis and prolonging antibody production [53], interleukin-12 (IL-12) that generates both efficient hu-moral and T-cell responses in combination with peptide ags [46], IL-15 able to enhance and sustain CD8

+ T-cell re-

sponses [46, 53], and Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), able to promote granulocyte and macrophage maturation and support mature immune system responses to ag [47]. The relatively short life of re-combinant cytokines constitutes the main drawback of these molecules; this problem is being currently overcome by cy-tokine encapsulation into liposomes or using cytokine ex-

pression vectors co-administered with DNA vaccines [53]. As already mentioned, some substances exhibit more than one enhancing activity. Identical result can be obtained by conjugating different adjuvants together thus amplifying their efficacy: Detox B, an oil droplet emulsion of mono-phosphoryl lipid A and mycobacteria [54], and ASO4 that contains aluminium hydroxide and MPL [47] are two repre-sentative examples. However, despite the high number of studies on these substances, at present, only two immu-nological adjuvants are approved for clinical use worldwide: aluminium-based salts and MF59, a squalene-oil/water emulsion [40, 52, 55], and the interest in this field is still growing.

VACCINES UNDERGOING PHASE II AND III CLINICAL TRIALS

Several phase II and III clinical trials have recently dem-onstrated the promising and the therapeutic potential of CVs (Table 1). In the next paragraphs, we review ongoing clinical research in the field, describing, in particular, open/recruiting phase II/III trials as well as preliminary data from studies only cited in conference proceedings or original manufac-tures’ websites. Throughout the text, references to ongoing clinical trials are cited by clinical trial number code, visible on www.clinicaltrial.gov.

Belagenpumatucel-L

Belagenpumatucel-L (Lucanix®

) is a vaccine developed by NovaRx and is composed by 4 different lung cancer cell lines (2 adenocarcinoma, 1 squamous, 1 large cell), geneti-cally modified to express an antisense DNA molecule, that binds the transforming growth factor beta (TGF- )-producing gene, preventing its expression. This CV has reached a phase III clinical trial recruiting stage III-IV non-small cell lung cancer (NSCLC) patients. The study started on July 2008 and is still ongoing, 700 patients will be en-rolled. This international multicentre study is based on the use of Lucanix as maintenance therapy for NSCLC patients who responded to or maintained a stable disease following one regimen of front-line platinum-based combination che-motherapy. The primary end point is to compare the overall survival (OS) of patients treated with Belagenpumatucel-L versus placebo. Secondary outcomes include the evaluation of progression-free survival (PFS), quality of life (QOL), time-to-progression (TTP), overall tumor response, rate of brain metastases and incidence of adverse events (NCT00676507). This new immune-based therapy moved to phase III after promising results obtained in a randomized phase II study of 75 patients with stage II/IV NSCLC. De-spite the heterogeneity of enrolled patients, safety profile and survival advantage derived from this vaccine permitted to receive a fast-track status by the FDA. The rate of serious adverse reactions was similar for the different dose cohorts. Pain, fatigue, respiratory problems and cough were the most frequent events among vaccinated patients [56]. Belagenpu-matucel-L has been tested even on advanced brain cancers in a phase I study reporting promising results and a phase II/III clinical trial is going to be activated also in glioma patients [57].

90 Current Cancer Drug Targets, 2011, Vol. 11, No. 1 Cecco et al.

BLP25 Liposomal Vaccine

BLP25 liposomal vaccine (Stimuvax®

), developed by Biomira and Merck KGaA, is constituted by a peptide frag-ment of the MUC-1 antigen, encapsulated in a liposomal delivery system. The vaccine target is mucin-1 (MUC-1), a protein antigen widely expressed by common cancers, mainly on the apical membrane of epithelial cells. Like other members of the mucin family, MUC-1 contributes to the protection and function of mucosal cells. The intracellular part of the protein may also participate in signal transduction pathways, through multiple interactions with intracellular

proteins. MUC-1 is over-expressed in the majority of epithe-lial cancers and even in some haematological malignancies. Merk KGaA is currently conducting two phase III trials based on Stimuvax.

BLP25 in Non-Small Cell Lung Cancer

The START (Stimulating Target Antigenic Responses To NSCLC) study is expected to include 1322 patients with un-resectable stage III NSCLC. Enrolled patients received concurrent or sequential chemotherapy/radiation and were randomized to maintenance vaccine or placebo in addition to best supportive care. Primary outcome was the comparison

Table 1. Cancer Vaccines: Targets and Stage of Development

Vaccine Target Indication Development Status Trial Reference

BELAGENPUMATUCEL-L

TGF- NSCLC

Glioma

III

I

NCT00676507

[57]

BLP25 MUC-1 NSCLC III NCT00409188

GSK1572932 MAGE-A3 NSCLC III NCT00480025

GV1001

Telomerase Pancreatic cancer

Liver cancer

NSCLC

III

II

I

NCT00425360

NCT00444782

NCT00509457

VITESPEN

Peptides-HSP Renal Cancer

Malignant melanoma

Colorectal cancer

Glioma

NSCLC

Withdrawal

III

II

II

II

[70]

NCT00039000

[69]

NCT00293423

NCT00098085

TG 4010 MUC-1 NSCLC III NCT00415818

MVA-5T4

5T4 Renal Cancer

Colorectal cancer

Prostate Cancer

III

II

II

NCT00397345

[73]

NCT00448409

Sipuleucel-T PAP (Prostate Acid Phospha-

te)

Prostate Cancer Preregistration NCT00065442

DC-Vax -Prostate

PSMA (Pros-tate Specific Membrane

Antigen)

Prostate Cancer III NCT00043212

CDX-110 EGFRvIII Glioblastoma Multiforme IIb NCT00458601

DC-Vax -Brain Tumor cell

lysate

Glioblastoma Multiforme II NCT00045968

V503 HPV Prevention cervi-

cal/vulvovaginal cancer

III NCT01047345

ZYC01a HPV-16 and

HPV-18 Human Papillomavirus-

associated dyplasias

IIb NCT00264732

DC-Vax -L Tumor cell

lysate

Ovarin Cancer I/II NCT00603460

PR1

MHC/proteina-se3

Acute myeloid leukaemia

Chronic myeloid leukae-

mia

Myelodysplastic syndro-mes

III

II

II

NCT00454168

NCT00499772

NCT00513578

BIOVAX-ID Idiotype vacci-

ne+GM-

CSF+HLM

Non-Hodgkin’s lymphoma III NCT00091676

Cancer Vaccines in Phase II/III Clinical Trials Current Cancer Drug Targets, 2011, Vol. 11, No. 1 91

of OS of all randomized patients; following randomization, patients in the investigational arm received a single intravenous infusion of cyclophosphamide, as immuno-adjuvant agent, three days before the first L-BLP25 vaccination. Patients then received eight consecutive, weekly subcutaneous vaccinations followed by maintenance injections every 6 weeks (NCT00409188). Common vac-cine-related adverse events were injection site reactions, fa-tigue and nausea [58].

BLP25 in Breast Cancer

The STRIDE (STimulating immune Response In aD-vanced breast cancer) study, which is expected to enrol 909 patients, primarily evaluates progression-free survival in recurrent or metastatic breast cancer patients with estrogen and/or progesterone receptor-positive non-resectable tumor (NCT00925548). At the end of March 2010, the company has temporarily suspended all recruiting Stimuvax trials worldwide, including START and STRIDE, as a prec-autionary measure due to an unexpected encephalitis in another Stimuvax trial.

BLP25 in Prostate Cancer

BLP25 liposomal vaccine has been tested in a pilot phase II study in hormone naïve men with biochemical failure after prostatectomy and little morbidity [59]. Even if this vaccine reduced prostate-specific antigen (PSA) levels, no further investigations are ongoing.

GSK 1572932A Recombinant Purified Protein

GSK 1572932A was investigated by GlaxoSmithKline in the MAGRIT (MAGE-A3 as Adjuvant Non-Small Cell Lung Cancer Immunotherapy) trial, which is expected to enrol approximately 2270 IB, II, o IIIA resectable NSCLC pa-tients, expressing MAGE-A3 (melanoma associated anti-gen). GSK 1572932A consists of a purified recombinant MAGE-A3 protein combined with ASCI (antigen-specific cancer immunotherapeutic), a novel class of compounds able to enhance the recognition and specific elimination of cancer cells by tumor-specific T cells. Expression of MAGE-A3 has been associated with a poor prognosis and is commonly de-tected in advanced cancer stages [60]. The MAGRIT trial (NCT00480025) is a randomised, double-blind study that aims to demonstrate immunotherapy-deriving benefits, in terms of disease-free survival (DFS). Both arms of recruited patients were vaccinated after surgery, but only one arm re-ceived additional chemotherapy. This trial also contributes to additional translational research aimed at identifying and validating a response predictive gene signature to therapy [61]. To date, the most common side effects are mild local (pain, redness, swelling) or systemic (fever, fatigue, muscle pain) reactions, observed within 24 hours from injection [62].

GV1001 Peptide Vaccine

GV1001, a peptide vaccine that targets the “universal an-tigen” telomerase, is being developed by Pharmexa. Telom-erase is a ribonucleoprotein complex highly expressed in almost all cancer types and is responsible for the unlimited dividing capacity of tumor cells [63]. The vaccine is based on immunogenic telomerase peptides that are expected to

prime patients T-cells, destroying cancerous cells via recog-nition of telomerase-expressing cells and leaving normal cells unharmed. GV1001 is undergoing clinical development also for hepatocellular carcinoma, NSCLC and pancreatic cancer, for which Pharmexa has been granted the orphan drug status in Europe and in USA. TeloVac, a phase III con-trolled trial (NCT00425360) will include 1110 patients with inoperable pancreatic cancer: patients are assigned to stan-dard chemotherapy (Gemcitabine plus Capecitabine) with concomitant GV1001 or chemotherapy with or without sub-sequent administration of GV1001. The primary endpoint will be survival, and then TTP and safety [64]. Results are expected by 2013. No toxicity or clinically severe adverse events related to the treatment have been observed so far [65]. GV1001 vaccine was previously tested in advanced hepatocellular and breast cancers with phase II trials, both stopped; on the other hand, a phase I study for advanced NSCLC showed the induction of GV1001-specific immune responses, associated with the induction of objective tumor responses [66].

Vitespen

Vitespen (Oncophage®

) is an autologous CV consisting of purified tumor-derived HSPs complexed with tumor ag peptides. Heat-shock proteins are highly conserved, stress-induced proteins with chaperone function for trafficking and delivering peptides within different cell compartments. These proteins can be included in vaccine formulations against malignancies [67]. Vitespen was submitted for mar-keting approval for the treatment of renal cell carcinoma in Europe and is currently used in a phase III clinical trial for malignant melanoma.

Vitespen in Melanoma

The main purpose of the C-100-21 Study Group was to assess the anti-tumor activity of Vitespen in patients with stage IV melanoma. Available data indicate similar survival rates between Vitespen and conventional chemo-immunotherapy, while a prolongation of median survival of 29% was observed in patients receiving at least 10 vaccine doses, compared to the conventionally treated arm. A high proportion of melanoma patients vaccinated with Vitespen remained disease-free at a median follow-up of 14 months [68], presenting only mild adverse events, including nausea, pyrexia, constipation, dyspnoea, arthralgia, back and ab-dominal pain.

Vitespen in other Cancers

Vitespen is also under development in patients with colo-rectal cancer, glioma and NSCLC. Different phase II studies were carried out on small patient series, but the results ob-tained so far were however useful to define the best candi-date population for confirmatory studies. A study involving 29 patients with metastatic colorectal cancer showed how Vitespen improved OS and DFS. Another phase II study achieved its primary goal by confirming the feasibility of deriving Vitespen from autologous tumor tissues in patients with non-resectable NSCLC (NCT00098085). Moreover, in March 2009, Antigenics announced Vitespen designation by the European Medicines Agency (EMEA) for orphan drug for the treatment of glioma (NCT00293423) [69].

92 Current Cancer Drug Targets, 2011, Vol. 11, No. 1 Cecco et al.

Vitespen in Renal Cancer

In October 2008, a marketing authorisation request was submitted to EMEA for the approval of early-stage renal cell carcinoma treatment with Vitespen, which however obtained a negative response, since the expected benefits of Vitespen did not outweigh the adverse effects in this cohort of cancer patients [70].

TG 4010 Recombinant Viral Vector

TG 4010 (MVA-MUC1-IL2), developed by Transgene, is a recombinant viral vector based on Ankara’s modified virus and DNA sequences, coding for the human IL-2 and the MUC-1 tumor antigen (MVA Modified Vaccinia An-kara-MUC1-IL2). The tumor-associated MUC-1 ag slightly differs from the wild type protein, and is also able to induce cellular anti-tumor immune response. MUC-1 is thus consid-ered as an attractive target for ag-specific cancer immuno-therapy and several MUC1-based therapeutic CVs are cur-rently under investigation in breast, renal, prostate cancer, and NSCLC.

TG 4010 in Breast Cancer

A phase II trial was conducted to assess the efficacy of TG 4010 as monotherapy in metastatic disease. Analysis of interim results showed some disease stabilization, but the rate of objective responses was not sufficient to justify the second step of the trial [71]. Thus, phase II clinical trials in prostate cancer and metastatic renal cell carcinoma patients were suspended in February 2005.

TG 4010 in Non-Small Cell Lung Cancer

A phase II randomized, open label trial was conducted in patients with stage IIIb/IV NSCLC. Primary aim of the study was to investigate the effect of TG 4010 in 2 arms of treat-ment: TG 4010 vaccination with concomitant (arm 1) or sub-sequent (arm 2) Vinorelbine and Cisplatin chemotherapy. In arm 1, 14 of 33 evaluable patients showed a partial response, whereas, in arm 2, the required number of responses was not reached and recruitment resulted discontinue at the end of the first stage. The OS measured at 12.7 and 14.9 months, respectively in arm 1 and 2, was similar to those reported in the literature with the same chemotherapy regimen used alone [72]. A further randomized phase IIb study (NCT00415818) assessed, in 148 patients, the efficacy of TG 4010 in combination with first-line chemotherapy (Cisplatin and Gemcitabine) compared to the chemotherapy regimen alone. The primary objective of the study in terms of PFS was met: all data confirmed the activation of the NK cells and the association with a favorable clinical outcome of NSCLC patients treated with TG 4010 and concomitant chemotherapy [73, 74]. The most frequent side effects were injection site reaction and asthenia. In December 2009, Transgene received FDA Fast Track status for TG 4010, after a phase III trial of TG 4010 in combination with first-line chemotherapy in patients with advanced NSCLC and normal levels of activated NK cells before treatment.

Mva-574

Oxford BioMedica and Sanofi-Aventis entered into a global licensing agreement to develop and commercialise

MVA 5T4 (TroVax®

) for the treatment and prevention of cancer. MVA 5T4 is a therapeutic vaccine targeting cells expressing the 5T4 antigen, expressed in a high proportion of different tumors (approximately 90% of renal cell tumors over-express this protein) but generally absent in normal adult tissues [75]. The product is based on an attenuated MVA, engineered to deliver the 5T4 ag and its expression induces a potent anti-tumor response. Clinical development of the vaccine is underway for renal cell cancer in Europe and USA, where Oxford BioMedica intends to seek the or-phan drug status, and for colorectal and prostate cancers.

Mva-5T4 in Metastatic Renal Cancer

Four phase II trials using this vaccine were completed,

including a total of 88 patients. The trials evaluated the

safety, immunogenicity and efficacy of TroVax either as a

single agent or in combination with standard therapy.

Although each study included different patient cohorts in

terms of prior treatment, concomitant therapy and tumor

histotype, the results indicated TroVax tolerability and its

ability to generate consistent immune responses to the 5T4

tumor ag (55 of 60 evaluable patients, 92%). TRIST

(TroVax®

Renal Immunotherapy Survival Trial) is a phase

III placebo-controlled trial designed to evaluate a minimum

of three doses of TroVax plus first-line standard therapy in

terms of prolonging survival in 733 patients with advanced

and metastatic renal cell carcinoma. Standard care therapies

for the treatment of this malignancy are IL-2, IFN- , or

Sunitinib (NCT00397345). In September 2009, the Company

announced interim results from the phase III TRIST: al-

though TroVax did not show a significant survival advantage

compared to placebo in the total population (median survival

of 20.1 months vs. 19.2 months; n=732; p=0.55), the survival

advantage was significant in one of the predefined subsets,

namely in patients with a good prognostic profile receiving IL-2 as standard of care (n=100; p=0.046).

Mva-5T4 in Metastatic Colorectal Cancer

Data from two single-arm, open-label, phase II trials,

undertaken to assess the safety and immunogenicity of

MVA-5T4 plus chemotherapy, showed that most of the

patients (9/11 and 10/12 of evaluable patients, respectively,

in these two trials) mounted an anti-tumor response with

exceptionally high numbers of specific CTLs [76-78].

Retrospective analysis on MVA-5T4 in colorectal cancer

patients showed a statistically significant relationship

between 5T4-specific antibody and favorable clinical

outcomes including TTP [79] or RECIST response score and

change in tumor burden [76]. Moreover, MVA 5T4 +/- GM-

CSF was associated with humoral and cell-mediated immune

responses in patients with metastatic hormone-refractory

prostate cancer in a phase II study enrolling 27 patients

(http://NCT00448409). Treatment combination showed

similar clinical and immunological responses compared to

MVA 5T4 alone: all 24 evaluable patients had robust

antibody responses against 5T4 and 9 patients also showed

strong 5T4-specific T-cell responses. Time to disease

progression, a secondary outcome, was significantly longer

in 5T4-specific T-cell responders compared with non-

responders. Intramuscular vaccine injections were well

Cancer Vaccines in Phase II/III Clinical Trials Current Cancer Drug Targets, 2011, Vol. 11, No. 1 93

tolerated and adverse events only included grade 1 fever and bone pain [80].

Sipuleucel-T

Sipuleucel-T (APC8015-PROVENGE ) is a DC-based vaccine, developed by Dendreon Corporation, for the treat-ment of androgen-independent prostate cancer (AIPC) and androgen-dependent prostate cancer (ADPC). This vaccine contains mature, autologous patient’s APCs obtained via standard leukapheresis procedure approximately two days before each infusion. Patient’s APCs were cultured with a recombinant fusion protein, the PA2024, consisting of prostatic acid phosphate (PAP) linked to GM-CSF. PAP is expressed in the majority of prostate cancers, while is absent in non-prostatic tissues, allowing Sipuleucel-T to stimulate tumor-specific immune responses, sparing hosts normal cells [81].

Sipuleucel-T in Androgen-Independent Prostate Cancer

Sipuleucel-T is in a late-stage development for the treat-ment of AIPC, with promising results derived from IMPACT (NCT00065442) and Pro-ACT (NCT00715078) trials. IMPACT (IMmunotherapy for Prostate AdenoCarcinoma Treatment) recruited 512 patients in a phase III, double blind trial and compared Sipuleucel-T versus placebo. Intention To Treat (ITT) analysis showed an advantage in the median survival (4.1 months) and an improvement in 3-year survival by 38% compared to placebo. Moreover, the IMPACT study demonstrated the ability of Sipuleucel-T to reduce the risk of death by 22.5% compared to placebo controls (HR=0.775) [82-84]. The most common grade 1 and 2 side effects asso-ciated with treatment were chills, pyrexia, headache, asthe-nia, dyspnoea, vomiting, and tremor. In April 2010 the FDA approved Sipuleucel-T for the treatment of asymptomatic or minimally symptomatic metastatic castration-resistant (hor-mone-refractory) prostate cancer.

Sipuleucel-T in Androgen-Dependent Prostate Cancer

Sipuleucel-T was investigated in two completed studies, PROTECT (PROvenge Trial of Early Prostate Cancer Ther-apy) (NCT00779402), and NCT00027599 (only defined by code). PROTECT was a randomized, phase III trial that en-rolled 176 non-metastatic ADPC patients with high levels of prostate-specific antigen (PSA) after radical prostatectomy. Sipuleucel-T induced long-term memory immune responses, maintained with subsequent boosting. Results also showed that CD54 up-regulation on APCs, a measure of the potency of the vaccine, correlates with the immune activation [85]. NCT00027599, a phase II trial, investigates Sipuleucel-T vaccination in combination with Bevacizumab and enrolled patients with progressive prostate cancer after radiation therapy and/or surgery. This combination treatment resulted in PSA value decline and reduction in PSA doubling time. Furthermore, immune modulation was demonstrated by in-duction of an immune response against PA2024. Despite these encouraging observations, implications for clinical practice were limited by the lack of concurrent control arms of either APC8015 or Bevacizumab monotherapy [86]. Con-sequently, a randomized trial of APC8015 with or without Bevacizumab, in patients with hormone-refractory prostate cancer, has been considered to further investigate the clinical

and immunological effects achieved with the addition of VEGF (Vascular Endothelial Growth Factor) blockade to APC8015.

DCVax

DCVax®

is a DC-based vaccine technology, originated

by Northwest Biotherapeutics, to develop autologous vac-

cines, called DCVax®

-Prostate, DCVax®

-Brain and

DCVax®

-L for the treatment of hormone-refractory prostate

cancer (HRPC), glioblastoma multiforme (GBM) and ovar-

ian cancer respectively. DCVax platform technology was

created using patient’s DCs and a device based on tangential

flow filtration for manufacturing DC from patient’s leu-

kapheresis. This DCs preparation is loaded, ex vivo, with

specific ag(s) or target protein(s) according to the type of

cancer to treat. DCVax product is an experimental autolo-

gous cellular therapy designed to activate a full immune re-

sponse consisting of both specific T cell and antibody re-

sponses against patient’s TAA. Such immune response may

be effective in delaying time to disease progression and may

prolong survival while maintaining good quality of life.

About clinical trials, DCVax products were investigated for HRPC, GBM, and ovarian cancer.

DCVax in Prostate Cancer

DCVax-Prostate consists of autologous DCs loaded with

the Prostate Specific Membrane Antigen (PSMA). PSMA is

a 750 amino acid protein distributed in prostate cancers and

endothelium of non-prostate cancers. Differences in the cy-

toplasmic (prostate and other cancers) versus plasma mem-

brane distribution (prostate cancer) of the staining were de-

scribed [87-89]. As a therapeutic target for immune attack,

PSMA was exploited by several innovative ways. A pivotal

DCVax-Prostate phase III study (NCT00043212) is termi-

nated in 2005 with no accrual or further data. In summary,

DCVax-Prostate met the basic developmental steps of proof

of concept feasibility and detectable immunogenicity, but a

randomized trial is needed to convincingly demonstrate its

efficacy on event-free survival or overall survival [90-92].

DCVax in Brain Cancer

DCVax-Brain had encouraging early-phase trial results.

Long-term data from phase I support the overall safety of

DCVax-Brain: side effects reported include skin reactions,

pain or itching at the injection site, nausea/vomiting, fatigue,

diarrhoea, and low-grade fever. Clinical data showed that

median survival time is 36.4 months (p= 0.0004) and pa-

tients striking delays in progression (recurrence) of their

cancer. Typically, in spite of surgical removal of the initial

tumor, as well as radiation and chemotherapy, GBM recur in

just 6.9 months. In contrast, after DCVax-Brain treatment

the median time to disease progression (recurrence) is 26.4

months [93]. It has been granted authorisation for limited

distribution in Switzerland and it is still underway a phase II

placebo control study (NCT00045968) that will enroll 141

patients with newly diagnosed GBM, treated with surgery,

radiation and chemotherapy. The primary endpoint of this

study is PFS and the secondary OS and TTP: estimated pri-mary completion date will be December 2011.

94 Current Cancer Drug Targets, 2011, Vol. 11, No. 1 Cecco et al.

DCVax-L in Ovarian Cancer

DCVax-L consists of an autologous DC vaccine loaded with autologous ovarian tumor cell lysate. DCVax-L is un-dergoing two phase I trials for recurrent ovarian or primary peritoneal cancer. UPCC 11807 (NCT00683241) is an open label, single group assignment trial aimed at determining the feasibility and safety of administering DCVax-L, intra-dermally combined with Bevacizumab and oral metronomic Cyclophospamide. The study is ongoing, but no longer recruiting participants. UPCC 01808 (NCT00603460) is a phase I/II study that will enroll patients previously vaccinated in the UPCC 11807 clinical study. The trial is not yet open for participant recruitment. Primary objective of the phase I study will be the feasibility and safety assessment of administering DCVax-L in combination with autologous T cells, primed ex vivo with the same vaccine, after lymphode-pletion induced by high-dose cyclophophamide/fludarabine. In the phase II, the study will also investigate the PFS at 6 months in patients treated with maintenance DCVax-L vac-cination and oral metronomic Cyclophospamide as well as in patients previously treated with ex vivo CD3/CD28-costimulated autologous peripheral blood T cells after lym-phodepletion.

CDX-110 (Rindopepimut)

CDX 110 is an immunotherapeutic molecule composed by the epidermal growth factor receptor variant III (EG-FRvIII), a peptide derived from a tumor-specific splice vari-ant of the epidermal growth factor receptor (EGFR). EG-FRvIII is the result of exon 1 joining to exon 8 in the EGFR gene and is one of the best clinically characterized alterna-tive splice variants whose expression is restricted to tumor cells. Unlike EGFR, indeed, EGFRvIII is not expressed at detectable levels in normal tissues and can directly lead to cancer through its oncogenic properties, providing a constant growth signal to tumor cells. CDX-110 is designed to induce a tumor-specific immune response against neoplastic cells expressing the cell-surface EGFRvIII protein as in GBM. Two phase IIa trials showed that CDX-110 was immuno-genic and prolonged survival in patients with newly diag-nosed glioblastoma. In ACTIVATE (A Complimentary Trial of an Immunotherapy Vaccine Against Tumor Specific EG-FRvIII) trial, 18 newly diagnosed EGFRvIII-positive GBM patients were treated with CDX-110 given intra-dermally with GM-CSF without concomitant DCs. Toxicity was minimal and there was no evidence of autoimmune reac-tions. Both humoral and cytotoxic EGFRvIII immune re-sponses were enhanced in patients vaccinated with CDX-110 and the median TTP was 14.2 months, which compared fa-vourably to a historical control group matched for entry cri-teria and failure to progress after radiation (6.3 months). The OS was 26 months compared to 15 months of the historical controls [94, 95]. The second phase IIa clinical trial was the ACT II: this study evaluated the effectiveness of vaccination in combination with standard of care Temozolomide (TMZ) chemotherapy. The vaccine was given in combination with concurrent daily TMZ in monthly cycles after completion of radiation. Patients were vaccinated on day 21 of each cycle until progression. All patients enrolled in ACT II vaccinated in coordination with monthly cycles of TMZ had a median TTP of 15.2 months versus 6.4 months for historical controls

and a median survival of 23.2 months versus 15.2 months for historical controls [96]. In April 2007, a phase IIb/III study with CDX-110, called ACT III (NCT00458601), was started to confirm results obtained in phase IIa trials. Enrolment of 60 patients was completed in November 2009 and the pri-mary outcome evaluation was made in April 2010. This non-randomized, open label study was designed to evaluate the clinical activity of CDX-110 vaccination when given with standard of care treatment (maintenance TMZ chemother-apy). Study treatment will be given until disease progression and patients will be followed for long-term survival informa-tion, while the efficacy will be measured by the PFS status at 5.5 months from the date of first dose.

In December 2007 and in January 2008, respectively, the CDX-110 achieved orphan drug status and Fast Track desig-nation in the USA for the treatment of EGFRvIII expressing GBM.

Human Papillomavirus Vaccine V503

Although the review mainly focused on therapeutic vac-cines, some new prophylactic cancer formulations are also provided to give a more complete overview of this emerging field. A new preventive vaccine, developed by Merck, against nine of the most harmful strains of HPV is under study for the prevention of cervical, vulvar or vaginal cancer. The intramuscular vaccine, a nine-valent called V503, con-sists of VLPs containing the structural proteins for nine dif-ferent HPV types. V503 is being compared with Gardasil

®, a

quadrivalent vaccine already on the market targeting the two most deadly HPV types. In September 2007, a phase III clinical trial has started with V503 administered to women aged 16-26. The trial is comparing the vaccine Gardasil

® and

approximately 14620 women are expected to be recruited (NCT00543543). This study will assess the safety, efficacy, and immunogenicity of V503 in comparison to Gardasil

®.

Primary endpoint is the evaluation of the incidence of HPV-related cervical, vaginal or vulvar disease. The experimental vaccine and the active comparator are administrated in three separate 0.5 ml intramuscular injection over a six month pe-riod. Merck expects to complete the study by June 2013 and to complete the evaluation of primary outcome by November 2011. In August 2009, another phase III study has started to evaluate the immunogenicity and tolerability of V503 in pre-adolescent subjects between 9 and 15 years old not sexually active, in comparison with young women 16 to 26 year olds. The estimated enrolment is 2800 patients and first data are expected by April 2011 (NCT00943722). Tolerability of V503 will be evaluated in girls and women between 12 and 26 years old, previously vaccinated with Gardasil

®, in a

placebo-controlled phase III study started in February 2010 and that will enroll 900 subjects (NCT01047345). Toxicity associated with the treatment may include injection site pain and swelling, pelvic discomfort during the exam, and/or vaginal bleeding in cases where a biopsy is needed for fur-ther examination.

ZYC01a (Amolimogene)

The vaccine is developed by MGI Pharma Biologics (Ei-sai) and is currently under investigation for the treatment of HPV-associated dysplasias, which can evolve to cancer if

Cancer Vaccines in Phase II/III Clinical Trials Current Cancer Drug Targets, 2011, Vol. 11, No. 1 95

untreated. ZYC01a bepiplasmid is a polymer-encapsulated DNA vaccine consisting of a plasmid expressing a chimeric peptide comprising immunogenic hybrid epitopes from HPV-16, and HPV-18 E6 and E7 proteins and HLA-DRalpha intracellular trafficking peptide [97]. The vaccine is intended to stimulate a long-lasting CTL response against HPV to eliminate the dysplasia. ZYC101a has been reported in phase I trials as an effective agent in the treatment of HPV-related lesions [98, 99]. Based on these findings, Gar-cia et al. [100] undertook a phase IIb trial of Amolimogene: a multicenter, placebo-controlled study, conducted in 161 patients with high-grade cervical-dysplasia (biopsy-confirmed CIN 2/3). Subjects were randomized into three groups, receiving three intramuscular doses of placebo, or 100 or 200 mcg of ZYC101a. The primary end-point of this study was histologically-confirmed resolution of cervical intraepithelial neoplasia (CIN) grade 2/3. Overall, high-grade cervical-dysplasia was resolved in 43% of patients treated with ZYC01a versus 27% of patients treated with placebo: this difference was not statistically significant. Interestingly, this potential benefit was much higher when evaluated in women younger than 25 years (70% and 23% respectively; p=0.007). The most common adverse events were moderate and generally limited to the injection site [100]. A multicen-tre phase II/III clinical trial (NCT00264732) is ongoing in 13 to 25 years old patients with high-grade cervical intraepithe-lial lesions of the uterine cervix (CIN 2/3): patients enrol-ment was completed in December 2009.

B Cell Lymphoma Vaccine BiovaxID®

BiovaxID®

is a personalized, patient-specific therapeutic vaccine targeting cancerous B-cells of relapsed or recurrent Non-Hodgkin’s Lymphoma (NHL). The vaccine consists of a lymphoma-associated, KLH-conjugated idiotypic ag and adjuvated with GM-CSF, essential for eliciting tumor-specific CD8 T-cell responses. Since NHL arises from a single B-cell, every lymphoma cell in patient’s body will express the same idiotype. BiovaxID uses this idiotype to generate a patient-specific vaccine that stimulates patient’s antitumor immune responses.

A phase II study, including 20 patients with follicular NHL in complete remission after chemotherapy, demonstrated efficacy of the vaccine in sustaining a complete molecular response and its tolerability [101]. In another phase II trial, a similar group of 26 patients received five cycles of BiovaxID monthly together with GM-CSF. Despite the lack of residual tumor B-cells as a result of che-motherapy and Rituximab treatment, antibody responses were detected in the majority of evaluable patients. CD4

+

and CD8+ T-cell responses against the vaccine occurred in

87 and 100% of treated patients, respectively, suggesting the involvement of professional APCs, such as DCs, in the ab-sence of B-cells. After 46 months median follow-up in 26 patients, OS was 89%, median event-free survival was 22 months and 19% of patients remained in continuous complete first remission [102]. The primary objective of the phase III study is to definitively confirm the safety and efficacy of BiovaxID as measured by a significant prolongation of DFS when administered to patients with indolent follicular NHL in their first complete remission

(NCT00091676). Final results from this study showed that the median duration of complete remission in the vaccine treated arm was 44.2 months, with a DFS of 30.6 months, representing a clinically and statistically significant result compared to the control arm. The vaccine prolonged DFS by 13.6 months with a median follow-up of 56.6 months. At 36 months, 61% of patients receiving the vaccine were disease-free, compared with 37% in the control arm [103].

PR1 Vaccine

PR1 is a nine amino acid, HLA-A*0201-restricted peptide, shared by two myeloid leukaemia-associated antigens, proteinase 3 (PR3) and neutrophil elastase (NE), over-expressed in myeloid leukemia cells. PR1 is able to induce powerful HLA-A*0201-restricted CD8

+ T-cell

responses that selectively kill myeloid leukemia cells. The detection of low frequencies of PR1-specific CD8

+ T cells in

patients with chronic myeloid leukemia and, at higher frequencies, in patients entering molecular remission after allogeneic stem cell transplantation supports the hypothesis of the existence of a natural immunity to PR1. This spontaneous response could be further boosted by vaccination to enhance immunity to leukemia [104].

The vaccine is under phase III development for the treatment of patients with acute myeloid leukaemia (AML) in remission. The placebo-controlled trial is assessing the vaccine mixed with montanide ISA-51 VG adjuvant and administered with GM-CSF in elderly patients with AML in first complete remission or in adults in second complete remission. Administration of vaccine therapy with GM-CSF may be an effective treatment for AML, although it still has to be elucidated whether this combination is more effective, in terms of overall survival, than giving placebo together with GM-CSF in treating AML (NCT00454168). Preliminary data from two phase II studies show that the new vaccine was generally well tolerated and toxicity was limited to grade 1-2 injection site reaction (NCT00499772, NCT00513578).

ADVANTAGES AND DRAWBACKS IN THE FIELD OF CVs

The therapeutic approach of CVs has several advantages if compared with conventional anti-cancer treatments, espe-cially if toxicity is considered. The ability of CVs to poten-tially circumvent drug cross-resistance and the persistence of the antitumor effect due to immunologic memory would ob-viate the requirement for prolonged, repetitive cycles of ther-apy [105, 106], thus reducing side effects. Moreover, the development of therapies specifically targeting TAA has the advantage to direct the immune response mainly against tu-mor cells and only few other normal tissues [107].

Some CVs are relatively simple to obtain, as it is the case of tumor-cell-based vaccines, which carry all the relevant tumor ags needed by the immune system to mount an effec-tive antitumor response, making it possible to develop vac-cines without knowing the specific ags expressed by a par-ticular tumor [107].

96 Current Cancer Drug Targets, 2011, Vol. 11, No. 1 Cecco et al.

Unfortunately, despite the promising results coming from the pre-clinical models, the first clinical data about CVs were quite disappointing. Several reasons may explain the limited clinical success of CVs, including the trial design, the spe-cific vaccination approach and host-related factors [108]. Probably, one of the main limitations has to be ascribed to the type of patients recruited in the various clinical studies: generally, phase I and some phase II clinical trials are per-formed on patients with advanced disease, after administra-tion of multiple chemotherapy regimens and often with se-vere immune-impairment. These limitations can be over-come by testing CVs in the setting of minimal residual dis-ease or in patients with complete remission after primary treatment [107]. Indeed, it has been shown that the interplay between host and tumor is fundamental for the success of CVs; through a selection process, the host may lead to the selective enrichment of clones of highly aggressive trans-formed cells, which are so undifferentiated that no longer express cancer cell specific molecules or express new cell surface ags [109]. In addition, immune tolerance is a typical ground of CVs failure. In fact, the down-regulation of the antigen processing machinery (TAA/HLA molecule), the production of putative immunosuppressive cytokines, the expression of lymphotoxic molecules (i.e. Fas ligand), and the lack of co-stimulatory molecules by tumor cells [55, 105, 109] favour the selection of highly resistant, poorly immu-nogenic, and aggressive clones of tumor cells [109]. Fur-thermore, only a few TAA probably behave as true tumor rejection ags, being the majority of TAA recognized as self molecules, and, as such, ignored by the immune system. Moreover, an immunosuppressive microenvironment fa-voured by tolerizing DCs and regulatory T cells can nega-tively contrast the antitumor immune response [105, 109].

Some other disadvantages have to be ascribed instead to the type of vaccine. Peptide vaccinations, for example, did not show clinical efficacy unless when combined with IL-2, often inducing only an irrelevant peptide-specific response [107]. On the other hand, the low yield of autologous tumor cells may limit the number of immunizations that can be given to patients, and potentially cause hyperstimulation of the immune system, with consequent autoimmunity effects. The lack of functional co-stimulatory molecules on tumor cells may also contribute to the anergic status of T cells [107, 109]. One of the main drawbacks of CVs is also the func-tional dissociation between systemic and local immune re-sponses: peptide-based vaccines can effectively generate a quantifiable T cell-specific immune response in the periph-eral blood of cancer patients, though such a response may be not related with a clinically evident tumor regression [110]. This is mainly linked to the lack of reliable immunologic surrogate markers of clinical response, one of the still imma-ture aspects of cancer immunotherapy. The main question in this respect is whether the measured immune response corre-lates with the antitumor activity of the vaccine. Antibody production has been repeatedly reported to correlate with clinical outcome [105], although in the case of the MUC-1 antigen for breast cancer treatment, antibody responses alone do not appear to be related with clinical response [106]. Findings regarding the diagnostic significance of T-cell re-sponses are conflicting, likely because no currently available in vitro assays can accurately mirror the in vivo antitumor activity of T-cells [55]. Several techniques have been tested

for this reason, but there is a critical need to validate these assays as surrogates for vaccine potency and clinical effects [111]. Enzyme-linked immunospot (ELISPOT) has the low-est limit of detection, but it is not a rapid assay, tetramer-based analysis yields quantitative but not functional data, intracellular cytokine flow cytometry [55] displays a less sensibility compared with ELISPOT, but offers the advan-tage of being faster and able to identify subsets of antigen-reactive cells [111]. Recently, improvements in optic and digital imaging have led to novel imaging techniques allow-ing the tracking of the migration of individual immune cells ex vivo and in vivo, and the detection of the dynamic interac-tions between T cells and APC or tumor cells within com-plex microenvironments, including lymphoid tissue and es-tablished tumors [112].

Finally, another important limitation of the modern knowledge about CVs is the lack of informative predictors of immunologic or clinical response, but some interesting ideas come from the HLA genotyping used to select patients po-tentially sensitive to HLA-specific peptide vaccines and the feasibility of defining tumor profiles as predictor of clinical response [113].

NEW TARGETS AND NEW METHODS AS FUTURE

PERSPECTIVES FOR IMPROVED CVs

To overcome all these drawbacks and make CVs a reli-able therapeutic approach for cancer, it is necessary to think about new target molecules and new methods that will in-clude most of the advantages of this treatment while obviat-ing to current limitations.

Generally, the main characteristics of the novel targets might be maximal immunogenicity, broad expression by different tumor types and maximal tumor specificity [55]. This profile identifies for example tumor angiogenesis-related ags, or the so-called universal tumor-ags, i.e. immu-nogenic molecules preferentially expressed by multiple tu-mor types. Candidate epitopes tested for their ability to elicit antigen-specific CTL responses from healthy donors and cancer patients ex vivo are survivin (an anti-apoptotic pro-tein) and telomerase [105]. On the other hand, a particular emphasis should be probably given to the exploitation of “unique” tumor ags, as they are considered the real media-tors of tumor rejection: these ags are extremely tumor spe-cific, lack any possible form of tolerance if compared with shared ags, are multiple-expressed by a single tumor and are resistant to host immunoselection, being usually essential to the maintenance of the malignant phenotype [114].

A particularly promising approach is the use of multipep-tide-based CVs, which may successfully bypass tumor het-erogeneity and the selection of ag-negative clones able to escape peptide-specific immune responses. In addition, sev-eral lines of evidence support the efficacy of combining HLA class I- and II-restricted epitopes, thus eliciting both CD4- and CD8-mediated immune recognition. These ap-proaches permits to target ags derived from different cell components of tumor microenvironment, thus broadening the spectrum of critical targets of CVs [115]. The design of poly- or multi-epitopic T cell vaccines implies that poorly immunogenic epitopes are identified, and that specific modi-fications are engineered to improve HLA binding or TCR

Cancer Vaccines in Phase II/III Clinical Trials Current Cancer Drug Targets, 2011, Vol. 11, No. 1 97

interaction and capitalize on the full diversity of the human T cell repertoire [107, 116, 117]. To this end, Synthetic Long Peptides (SLPs) have evolved as a simple solution to many intrinsic problems of single peptide vaccines. SLPs are not able to bind directly to MHC class-I: they are primarily processed and presented by professional APC and therefore stimulate CTL predominantly within the strong environment of the inflamed draining lymph node. Moreover, the in-creased duration of in vivo epitope presentation of a SLP vaccine, favours the clonal expansion of effector T cells and thereby their IFN- production. Consequently, HLA-restriction is no longer an obstacle since patient’s DCs select by themselves the appropriate CD4 and CD8 T-cell epitopes [55, 118]. “Bivalent” peptides able to bind both HLA class I and class II molecules, have considered as a promising solu-tion too, because of the crucial role of helper T lymphocyte in modulating the magnitude and durability of CTL response [55]. Finally, Bettahi and colleagues proposed a self-adjuvanting glyco-lipopeptide vaccine bearing B cell, CD4

+

and CD8+

T cell epitopes to ensure a whole immune response [119].

However, as single treatment modalities, current CVs are not probably endowed with the potency required to over-come the obstacles of tumor burden and immune tolerance in patients with established cancer [98]. For this reason, the combination of CVs with traditional therapies (monoclonal antibodies and chemotherapy in particular) seems to be the most accomplishing and rational way of success.

Monoclonal antibodies (mAb) are therapeutic reagents that not only provide passive immunotherapy through anti-body dependent cell-cytotoxicity (ADCC), but can also pro-mote active immunity resulting in augmentation of immu-nologic memory. There is preclinical and clinical evidence demonstrating the successful combination of mAb with CVs. Trastuzumab (Herceptin, a mAb against HER-2/neu), for example, can enhance the lytic activity of MHC class-I re-stricted HER-2/neu specific CTL, besides recruiting innate immune effectors and sensitizing the tumor cells to apoptosis [106, 120]. In vivo antibody-mediated blockade of CTLA-4 (Cytotoxic T Lymphocyte Antigen 4, an inhibitor receptor) with the mAb ipilimumab, potentiates T cell response to poorly immunogenic tumors in murine models and in pa-tients with prostate or melanoma cancer [121]. Ipilimumab in combination with a whole tumor-cell vaccine also showed promising preliminary results in patients with asymptomatic androgen-independent prostate cancer, and when combined with a peptide vaccine induced antitumor activity in patients with melanoma (www.asco.org

1)[122]. CD40-specific ago-

nist antibodies (the CD40/CD40L pathway plays a central role in the regulation of humoral and cellular immunity) can prevent tumor-induced T cell tolerance and break established T cell tolerance, augmenting the efficacy of CVs. Moreover, combining an agonist OX40 antibody (OX40 is a member of the tumor necrosis factor receptor superfamily expressed transiently on activated CD4

+ T cells) and the adoptive trans-

fer of tumor-specific T cells showed a greater antitumor ef-fect than the infusion of T cells alone. OX40 engagement during primary immunization breaks peripheral CD4

+ T cell

tolerance, increasing the survival of memory T cells [105].

1ASCO 2006 Annual Meeting Summaries, Abstract n. 2500.

Chemotherapeutic agents are commonly used for their overt cytotoxic effects; however some of them can also ei-ther enhance or inhibit antigen-specific immune response depending on dose and timing of administration in relation to antigen exposure [105, 106, 123]. Some drugs are able to promote T helper 1 inflammatory cytokine producing cells (paclitaxel, cyclophosphamide) or enhance NK cell reactivity (bortezomib). Other drugs can block suppressor cells: gemicitabine blocks myeloid derived suppressor cells (MDSC), whereas bortezomib and cyclophosphamide ham-per regulatory T cells (Treg) [124]. There are chemothera-peutic drugs that can render tumor cells more susceptible to immune destruction: azacytidine upregulates MHC-I mole-cules; topoisomerase enhances susceptibility to NK cell cy-totoxicity; mitomycin, 5-fluorouracil, doxorubicin and cis-platin increase expression of death receptor [106, 124]. Fi-nally, several drugs such as doxorubicin, mitomycin, cyclo-phosphamide, vincristine, and methotrexate can enhance macrophage-dependent killing [106, 124, 125].

Although some of these chemotherapeutics had already been tested in clinical trials for their ability to influence the immune response to CVs, the design of effective combina-tion therapies needs further analysis of fundamental parame-ters, such as the most appropriate dose of the chemothera-peutics: doxorubicin, low doses, plus GM-CSF-secreting CT-26 as colon cancer vaccine [98], cyclophosphamide or paclitaxel, low doses, plus GM-CSF in breast cancer [126, 127]. In addition to dosage issues, the time of administration of the chemotherapy is also critical: cyclophosphamide can overcome natural and acquired immune tolerance if given before ag exposure, but it promotes the induction of immune tolerance if given concomitantly with the ag [128]. On the other hand, in vitro assays gave also interesting results: for example the up-regulation of MHC class I and cancer testis ags by pre-treating tumor cells with the de-methylating agent 5-aza-2’-Deoxycytidine can restore melanoma- and renal cell carcinoma-specific CTL activity [129].

Finally, it has been suggested that also the lymphopenia induced by chemotherapeutic agents could play a role in favouring the immune response to CVs. Immune manipula-tion by active immunization or the adoptive transfer of TAA-specific T cells during the period of immune reconstitution after ablative treatments, might favour the development of a T cell repertoire skewed toward a desired antitumor specific-ity [99, 117]. Two recent trials testing the adoptive transfer of tumor-specific lymphocytes in the setting of non-myeloablative chemotherapy for melanoma suggest that this approach may result in clinically relevant effects [130, 131].

Interestingly, also vaccination against TAA is thought to sensitize the tumor against subsequent chemotherapeutic treatments [120]. Therefore, elucidation of the interactions between established cancer therapies and experimental vac-cines should facilitate the rational integration of CV within innovative combination regimens characterized by enhanced clinical efficacy [106].

CONCLUDING REMARKS

The most promising avenue of CVs research will lead to a better understanding of the mechanisms underlying the

98 Current Cancer Drug Targets, 2011, Vol. 11, No. 1 Cecco et al.

complex interactions between the immune system and cancer cells. Greater importance has to be attributed to the link be-tween innate and adaptive immune responses, as well as to the growing evidence that CTL, T helper cells, and B-cells can synergistically concur to determine the immune rejection of cancer [55]. These studies will lead to the development of improved CVs with enhanced clinical efficacy. At present, however, available CVs probably have a role as adjuvant to both traditional (radiation and chemotherapeutic) therapies and in the management of minimal residual disease follow-ing surgical resection of the primary tumor [109]. In this setting, CVs may be included among the adjuvant strategies for patients who are at high risk of relapse [106]. In perspec-tive, it will be of relevance to go beyond the use of CVs mainly limited to patients with advanced diseases, often after having exploited all traditional therapeutic modalities [109]. Clinical studies aimed at evaluating the clinical efficacy of current CVs in the adjuvant setting will allow a more thor-ough assessment of the real therapeutic potential of this in-novative immunotherapeutic approach.

AKNOWLEDGEMENT

This work was supported by Department for Clinical and Supportive Care and Medical Oncology Department of the “Centro di Riferimento Oncologico, CRO-IRCCS”, Aviano, Italy.

ABBREVIATIONS

ADCC = antibody dependent cell-cytotoxicity

ADPC = androgen-dipendent prostate cancer

AIPC = androgen- independent prostate cancer

AML = acute myeloid leukaemia

ag = Antigen

APC = antigen-presenting cell

ASCI = Antigen-specific cancer immunotherapeutic

BCG = bacillus Calmette-Guerin

CIN = cervical intraephitelial neoplasia

CTL = cytotoxic T lymphocytes

CTLA-4 = cytotoxic T lymphocyte antigen 4

CV = cancer vaccine

DC = dendritic cell

DSF = Disease free survival

EGFR = epidermal growth factor receptor

EGFRvIII = epidermal growth factor receptor variant

EMEA = European Medicines Agency

FCA = Freund’s complete adjuvant

FDA = Food and Drug Administration

GBM = glioblastoma multiforme

GM-CSF = granulocyte-macrophage colony-stimulating factor

KLH = keyhole limpet haemocyanin

HCC = hepatocellular carcinoma

HLA = human leukocyte antigen

HBsAg = hepatitis B surface antigen

HBV = hepatitis B virus

HPV = human papillomavirus

HRPC = hormone refractory prostate cancer

HSP = heat-shock proteins

IL = interleukin

IFN = interferon

ITT = intention-to-treat

mAb = monoclonal antibody

MAGE A3 = melanoma antigen-A3

MDSC = myeloid derived suppressor cells

MHC = major histocompatibility complex

MPL = monophosphoryl lipid

MUC1 = mucin-1

NE = Neutrophil elastase

NHL = Non-Hodgkin’s lymphoma

NK = natural killer

NSCLC = non-small cell lung cancer

ODN = oligodeoxinucleotide

OS = overall survival

PAMP = pathogen-associated microbial patterns

PAP = prostatic acid phosphate

PFS = progression free sulrvival

PSA = prostate-specific antigen

PR3 = Proteinase 3

PSMA = prostate specific membrane antigen

QOL = quality of life

RECIST = Response Evaluation Criteria In Solid Tu-mors

SLP = Synthetic Long Peptides

TAA = tumor-associated antigen

TCR = T cell receptor

TGF- = Transforming growth factor beta

TLR = toll-like receptor

TMZ = temozolomide

Treg = regulatory T cells

TSA = tumor-specific antigen

TTP = time to progression

USA = United States of America

VEGF = Vascular Endothelial Growth Factor

VLP = virus-like particles

Cancer Vaccines in Phase II/III Clinical Trials Current Cancer Drug Targets, 2011, Vol. 11, No. 1 99

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Received: February 19, 2010 Revised: May 31, 2010 Accepted: June 17, 2010

PMID: 21062241