Carcinoembryonic Antigen (CEA)-Based Cancer Vaccines: Recent Patents and Antitumor Effects from Experimental Models to Clinical Trials

Recent Patents on Anti-Cancer Drug Discovery, 2012, 7, 265-296 265 Carcinoembryonic Antigen (CEA)-Based Cancer Vaccines: Recent Patents and Antitumor Effects from Experimental Models to Clinical Trials

Mario Turriziania,#, Massimo Fantinib,#, Monica Benvenutob,#, Valerio Izzib, Laura Masuellic, Pamela Sacchettic, Andrea Modestib and Roberto Beib,* aDepartment of Internal Medicine, University of Rome “Tor Vergata”, 00133, Rome, Italy; bDepartment of Clinical Sci- ences and Translational Medicine, University of Rome “Tor Vergata”, 00133, Rome, Italy; cDepartment of Experimen- tal Medicine, University of Rome “Sapienza”, 00161, Rome, Italy

Received: April 24, 2012; Accepted: May 9, 2012; Revised: May 18, 2012

Abstract: Carcinoembryonic antigen (CEA), a glycosylated protein of MW 180 kDa, is overexpressed in a wide range of human carcinomas, including colorectal, gastric, pancreatic, non-small cell lung and breast carcinomas. Accordingly, CEA is one of several oncofetal antigens that may serve as a target for active anti-cancer specific immunotherapy. Ex- perimental results obtained by employing animal models have supported the design of clinical trials using a CEA-based vaccine for the treatment of different types of human cancers. This review reports findings from experimental models and clinical evidence on the use of a CEA-based vaccine for the treatment of cancer patients. Among the diverse CEA-based cancer vaccines, DCs- and recombinant viruses-based vaccines seem the most valid. However, although vaccination was shown to induce a strong immune response to CEA, resulting in a delay in tumor progression and prolonged survival in some cancer patients, it failed to eradicate the tumor in most cases, owing partly to the negative effect exerted by the tumor microenvironment on immune response. Thus, in order to develop more efficient and effective cancer vaccines, it is necessary to design new clinical trials combining cancer vaccines with chemotherapy, radiotherapy and drugs which target those factors responsible for immunosuppression of immune cells. This review also discusses relevant patents relating to the use of CEA as a cancer vaccine. Keywords: Cancer, CEA, CTL, immunotherapy, tumor antigen, vaccine.

INTRODUCTION The goal of cancer immunotherapy is to disrupt the immune tolerance to tumor-specific antigens (TSAs) and tumor Carcinoembryonic antigen (CEA) is a glycosylated associated antigens (TAAs), thus inducing a strong immune membrane-bound protein of 180 kDa expressed in a high response to these antigens expressed by cancer cells. In this percentage of several human carcinomas, including colorec- regard, it was demonstrated that tolerance to CEA could be tal, gastric, pancreatic (90%), non-small cell lung (70%) and interrupted by immunizing mice with recombinant human breast carcinomas (50%) [1]. CEA [10] without leading to autoimmune disease [11, 12]. The high expression of CEA by tumor cells and its secre- This review will discuss the use of CEA as a target for tion into the serum make this antigen suitable for use as a cancer immunotherapy both in animal models and clinical tumor marker, especially in the staging and follow-up of trials and provide information on novel, patented CEA-based patients with colorectal cancer [2, 3]. CEA is also expressed vaccines formulated to improve its immunogenicity and its as an oncofetal antigen during fetal development and, at low antitumor activity. levels, in normal colonic epithelial cells. Furthermore, CEA may have different molecular weights in normal vs. cancer Thus, building off of the encouraging results obtained cells due to a different N-glycosylation pattern in cancer from immunotherapy protocols provided in animal models cells [4]. and clinical trials, it would be of great interest to develop new therapies to treat tumors that express CEA. CEA is a self-antigen and although abundantly expressed on the surface of cancer cells, it induces weak spontaneous immune responses in cancer patients. However, autoantibod- CEA-BASED ANTI-CANCER VACCINES: MOUSE MODELS AND RELATED PATENTS ies against CEA were detected in the sera of patients with cancers expressing CEA [5-9]. Several studies have shown that CEA can be envisaged as a target for cancer immunotherapy protocols. It was previously demonstrated that tolerance to CEA could be inter- *Address correspondence to this author at the Department of Clinical Sci- rupted by mouse immunization with recombinant human ences and Translational Medicine “Tor Vergata”, Via Montpellier 1, 00133 CEA [10] without leading to autoimmune disease [11, 12]. Rome, Italy; Tel: +39-06-72596522; Fax: +39-06-72596506; E-mail: [email protected] Since then, different modalities of immunization and # These authors contributed equally to this work experimental models to determine whether CEA immuniza-

2212-3970/12 $100.00+.00 © 2012 Bentham Science Publishers 266 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al. tion could induce an immune response capable of inhibiting (LS174T) expressing CEA efficiently counteracted tumor the growth of tumors expressing CEA have been employed. growth [21]. Examples of CEA-based vaccines using mouse models are Similarly, Johansonn et al. immunized C57BL/6 mice reported in Table 1. with a plasmid encoding the wild type form of human CEA Vaccination with viral or yeast vectors expressing CEA, (p91023B) and with pKCEA66 to better understand how the dendritic cells (DCs) pulsed with CEA-peptides, CEA- cellular localization of CEA could influence the generation plasmid DNA or with recombinant CEA has been proven to of the immune response. The authors confirmed the findings be successful in inducing a potent anti-tumor immunity in of Lund et al., but they also observed that mice immunized animal models [13-17]. with the p91023B showed higher titers of anti-CEA IgG than In addition, to enhance the potency of vaccination, com- mice immunized with pKCEA66. Moreover, the boost with recombinant CEA (rCEA) induced a significant increase in bined vaccination protocols have been employed as well. the humoral anti-CEA response in all mice [22]. This phenomenon was believed to be due to the plasma membrane DNA-based Vaccines Alone or Vaccines Plus Immu- localization of p91023B, confirming the hypothesis that the nostimulants secreted antigen is more efficient in inducing an antibody DNA vaccines have been shown to induce both humoral response than the intracellular antigen, which could instead and cell-mediated immune responses against CEA. Conry enhance a cell-mediated response [23]. et al. demonstrated that a polynucleotide vaccine composed To evaluate whether the anti-CEA response was protec- of the cDNA encoding human CEA was able to induce a tive against tumor growth, SCID mice were transplanted humoral and cell-mediated immune response in mice and with splenocytes containing NK and T cells derived from protect mice against challenge with mouse colon carcinoma immunized mice and then challenged with human CEA- cells transduced with human CEA (MC-38-CEA-2) [18, 19]. producing cells. The authors observed that SCID mice were Similarly, Luo et al. employed a plasmid DNA encoding protected against tumor challenge and hypothesized that NK human CEA alone or CEA adsorbed onto cationic micropar- and T cells could play a role in counteracting tumor growth ticles to determine whether CEA-induced tumor immunity [22]. was able to protect CEA transgenic mice against a lethal In addition, a DNA vaccine expressing a triple repeated challenge with MC38-CEA cells. The authors demonstrated CEA peptide (triCEA625-667) was used to immunize BALB/c that the CEA-based DNA vaccine formulated into cationic mice intramuscularly. After immunization, strong antigen- microparticles induced a more potent protective immunity specific T cell proliferation and elevated levels of anti-CEA than the naked CEA-based DNA vaccine. In addition, it was specific antibodies have been observed [24]. reported that intramuscular injection of a plasmid encoding murine granulocyte/macrophage colony-stimulating factor Novel CEA-like polynucleotides, encoding novel poly- (mGM-CSF) three days after immunization with the CEA- peptides with biological activity, isolated from a cDNA li- based DNA vaccine resulted in a marked increase in tumor brary have been disclosed in a US patent application. protective immunity and a reduction in tumor volume [20]. Moreover, expression vectors and cells genetically engineered to contain these polynucleotides have been reported. A new strategy to enhance the potency of the DNA plas- In particular, it was suggested that these polynucleotides and mid encoding human CEA involved the development of new polypeptides could be administered in combination with a plasmids encoding partial sequences of human CEA. Lund pharmaceutically acceptable carrier in the treatment of et al. employed a plasmid encoding a truncated form of hu- breast, prostate and colon cancer [25]. man CEA and a plasmid encoding the wild type form of human CEA. Based on evidence that the truncated CEA protein Similarly, improved CEA vector nucleotides useful for was retained in the cytoplasm of cells while the wild type treating and/or preventing cancer have been described by CEA protein was expressed on the membrane, the authors Parrington et al. in the US patent application hypothesized that the retention of the truncated CEA in the US20100285065. Inventors provided these improved vectors cells could enhance the presentation of CEA peptides in as- for using as cancer vaccine. Moreover, the inventors describe sociation with MHC class I molecules, thus improving cellu- that the pharmaceutical compounds, which contain a CEA- lar immune responses against CEA. Conversely, they hy- nucleic acid or -polypeptide, in combination with an accept- pothesized that the presence of the wild type CEA on the able carrier, can be administered orally, parenterally, by in- plasma membrane could enhance the humoral response. In halation spray, rectally, intranodally, topically and intratu- this regard, a plasmid was developed that encoded the trun- morally to treat mammals with tumors-expressing CEA [26]. cated human CEA form fused to a tetanus toxoid epitope The oral administration of a CEA-based DNA vaccine (pKCEA66). This plasmid was then used to intramuscularly was also employed to induce an efficient tumor-protective immunize C57BL/6 mice. The authors observed that mice immunity against colon cancer. Xiang et al. developed oral immunized with pKCEA66 developed a stronger IgG1- and CEA-based DNA vaccines that were effective in breaking cell-mediated response than mice immunized with the wild peripheral T cell tolerance against human CEA and inducing type CEA plasmid. In addition, the authors observed that 6 tumor-protective immunity in CEA-transgenic C57BL/6J out of 10 SCID mice that were transplanted with spleen cells mice. These vaccines led to the activation of CD8+ T cells derived from the C57BL/6 mice immunized with pKCEA66 and DCs, which were able to protect mice after a lethal chal- and then challenged with human colon carcinoma cells lenge with the murine MC38 colon carcinoma cell line trans-

CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 267

Table 1. CEA-based Cancer Vaccine in Mouse Models.

Type of Vaccine Mouse Model Effector Anti-Tumoral Effect Reference

DNA-BASED VACCINES ALONE OR VACCINES PLUS IMMUNOSTIMULANTS

hCEA/DNA (PcDNA3 plasmid encoding Syngeneic CEA-transduced colon CTLs, CD4+T Suppression of tumor [19] hCEA) carcinoma cells in C57BL/6 mice cells, antibodies growth (100%)

Reduced tumor volume Prime (hCEA/DNA)-boost (GM-CSF/DNA) MC38-CEA in CEA-tg C57BL/6 mice CTLs [20] (50%)

SCID mice transplanted with spleen cells IgG, IFN-, T hCEA/DNA (pKCEA66 plasmid encoding a from immunized C57BL/6 mice and Suppression of tumor cell responses, [21, 22] truncated hCEA form) inoculated with hCEA expressing cells growth (60%) NK (LS174T)

CTLs, CD4+T cells, Significant protective hCEA/DNA (triple repeated (CEA) ) BALB/c mice [24] 625-667 antibodies, immunity IFN-

Rejection of tumor hCEA/DNA (pCD40LT-CEA) MC38-CEA in CEA-tg C57BL/6J mice CTLs, DCs [27] challenge (50%)

CEA/KSA-positive Lewis lung carcinoma Reduction of tumor hCEA/DNA (pW-CEA) (LLC-CEA-KSA) cells in C57BL/6J CEA CTLs, DCs [28] growth (25%) tg mice

MC38-CEA-A2Kb in C57BL/6-CEA- hCEA/DNA (pHI-691DNA minigene) CTLs Tumor rejection (30%) [32] A2Kb double tg mice

hCEA/DNA (pCMV-CEA) plus IL-12/DNA Antibodies, Suppression of tumor CEA/LLC* cells in C57BL/6 mice [33] (VR-IL-12) CTLs, IFN- growth (80%)

hCEA/DNA (pCMV-CEA) plus IL-12/DNA Antibodies, Reduction of tumor CEA/LLC cells in C57BL/6 mice [34] (VR-IL-12) membrane bound CTLs, IFN- incidence (40%)

hCEA/DNA (pCMV-CEA) plus IL-4/DNA Antibodies, Reduction of tumor CEA/LLC cells in C57BL/6 mice [34] (VR-IL-4) membrane bound CTLs, IFN- incidence (80%)

hCEA/DNA (VR-CEA) plus B7.1/IgG/DNA Suppression of tumor CEA/LLC cells in C57BL/6 mice CTLs , IFN- [35] (VR-B7.1/IgG) growth (50%)

hCEA/DNA (VR-CEA) plus B7-1/Ig/DNA Reduction of tumor CEA/LLC cells in C57BL/6 mice CTLs , IFN- [36] (VR-B7-1wa/Ig) incidence (60%)

hCEA/DNA (pCEA) plus mGM-CSF/DNA IgG1, IFN-, T Suppression of tumor MC38-CEA-2 in C57BL/6 mice [37] (pGM-CSF) cell responses growth (80%)

ANTI-IDIOTYPE ANTIBODIES-BASED

VACCINES

CTLs, CD4+T Prolongation of survival 3H1 antibody MC38-CEA in C57BL/6 mice [40] cells, antibodies time (90% of mice)

CTLs, CD4+T Prolongation of survival 3H1 antibody plus CpG-ODN (1826) MC38-CEA in C57BL/6 mice [41] cells, antibodies time (100% of mice)

CEA-transfected C15 cells in C57BL/6J- Antibodies, Prolongation of survival 3H1 antibody plus CpG-ODN (1826) [42] TgN (CEAGe) 18FJP mice CTLs time (33%)

268 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al.

(Table 1) Contd….

Type of Vaccine Mouse Model Effector Anti-Tumoral Effect Reference

DC-BASED VACCINES

Reduction of tumor SW480 human CEA+ /HLA-A2.1+ colon growth (100%), CEA-Hsp70L1/DC CTLs [54] carcinoma cells in C57BL/6 nu/nu mice prolongation of survival time (37,5%)

Prolongation of survival Hiv-Tat/hCEA/DC MC38-CEA-2 cells in B6 mice CTLs [55] time (80%)

Inhibition of tumor CD4+T cells growth, prolongation of CRT-TAT-CEA/DC MC38-CEA-2 cells in C57BL/6 mice [57] and CTLs survival time (70 vs 40 days in control group)

Antibodies, CEA-transfected C15 tumor cells in CEA Rejection of tumor 3H1/DC CD4+T cells, [60] tg mice challenge (100%) CTLs

Regression of tumor MC-38-CEA-A2Kb cells in C57BL/6J- CD4+T cells growth (50% with 3H1/DC plus CTL peptides of CEA/DC [61] CEA-A2Kb mice and CTLs peptide-2; 67,5% with peptide-5)

Increasing of tumor free 3H1/DC plus CTL peptides of CEA/DC plus MC-38-CEA-A2Kb cells in C57BL/6J- CD4+T cells, survival (67% with [62] CpG-ODN CEA-A2Kb mice NK cells, CTLs peptide-1; 76% with peptide 2)

3H1/DC plus CTL peptides of CEA/DC plus MC-38-CEA-A2Kb cells in C57BL/6J- Suppression of tumor CTLs [63] anti-CD25 and anti-CTLA-4 antibodies CEA-A2Kb mice growth (90%)

CD4+T cells Suppression of tumor AdCEA/DC MC38-CEA-2 cells in C57BL/6 mice [64] and CTLs growth (100%)

MC38-CEA cells in C57BL/6J-TgN CD4+T cells, Suppression of tumor AdCEA-GM-CSF-IL-12/DC [66] (CEA Ge) 18FJP NK cells, CTLs growth (80%)

Reduction of tumor vo- AdCEA-AdSVV/DC plus CAR-TAT MC38-CEA-2 cells in C57BL/6 mice CTLs [67] lume

RECOMBINANT VECTOR-BASED

VACCINES

Suppression of tumor rV-CEA plus rV-B7 MC38-CEA-2 cells in C57BL/6 mice CTLs [71] growth (80%)

CTLs, CD4+T Regression of tumor rV-CEA plus IL-2 MC38-CEA-2 cells in C57BL/6 mice [74] cells growth (60-70% of mice )

Prolongation of survival rV-CEA/TRICOM plus GM-CSF and IL-2 MC38-CEA cells in CEA tg mice CTLs [76] time (83% of mice)

Prime (rV-CEA/TRICOM with rGM-CSF)- CTLs , CD4+T Prolongation of survival MC38-CEA cells in CEA tg mice [77] boost (rF-CEA/TRICOM plus rF-GM-CSF) cells time (56,3% of the mice)

Subcutaneous prime (rV-CEA/TRICOM plus rF-GM-CSF)-intratumoral boost (rF- Regression of tumor MC38-CEA cells in CEA tg mice CTLs [80] CEA/TRICOM plus rF-IL-2 and rF-GM- growth (80% of mice) CSF)

Significant decrease in CTLs , CD4+T rV-CEA/TRICOM plus rV-4-1BBL MC38-CEA cells in CEA tg mice tumor growth (2/15 mice [81] cells tumor free)

CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 269

(Table 1) Contd….

Type of Vaccine Mouse Model Effector Anti-Tumoral Effect Reference

Prime (rV-CEA/TRICOM plus mAb 9H10)- Suppression of tumor MC38-CEA cells in CEA tg mice CTLs [83] boost (rF-CEA/TRICOM) growth (20%)

Prolongation of survival Prime (rV-CEA/TRICOM followed by Y- CTLs, CD4+T time (50% of mice after 90-labeled anti-CEA mAb COL-1)-boost MC38-CEA cells in CEA tg mice [84] cells 50 days; 22% of mice (rF-CEA/TRICOM) after 50 days)

NK cells and Tumor growth inhibition rV/F-CEA/TRICOM plus IFN- MC38-CEA cells in CEA tg mice [86] CTLs and improved survival

Prime (rMVA-CEA/TRICOM)-boost (rF- CTLs, CD4+T Regression of tumor MC38-CEA cells in CEA tg mice [87, 88] CEA/TRICOM) cells growth

Suppression of tumor Prime (rV-CEA)-boost (ALVAC-CEA) MC38-CEA cells in CEA tg mice CTLs [89] growth (63% of mice)

Suppression of tumor ALVAC-CEA plus ALVAC-GM-CSF MC38-CEA-2 cells in CEA tg mice CTLs, IgG [90] growth (100%)

CD4+T cells, IgG, Suppression of tumor ALVAC-CEA MC38-CEA cells in C57BL/6 mice [91] IFN- ,IL-5 growth (100%)

Regression of tumor Ad5 [E1-, E2b-]-CEA MC38-CEA-2 cells in C57BL/6 mice IFN- and IL-2 [95] growth (100% of mice )

Prolongation of tumor- CEA-rAAV plus pNGVL3 MC38-CEA-2 cells in C57BL/6 mice DCs and IFN- free survival time (80% of [98] mice)

CTLs, antibodies, Regression of tumor PV-CEA RMA-CEAFL cells in CEA tg mice [101] IFN- growth (100% of mice)

U87 glioblastoma cells in BALB/c nude Regression of tumor MV-CEA Antibodies [102] mice growth (87% of mice )

CTLs, CD4+T Bac-CEA BALB/c mice N.A. [103] cells, NK cells

Prolongation of survival CTLs, CD4+T time, Yeast-CEA MC38-CEA-2 cells in CEA tg mice [108] cells reduction of tumor volume

Prime (rV/-CEA/TRICOM)-boost (yeast- Reduction in pulmonary LL2-CEA tumor cells in CEA tg mice CTLs [110] CEA) metastasis

PROTEIN- AND PEPTIDES-BASED

VACCINES

Antibodies, T-cell Protection from tumor bV-CEA/DOTAP MC38-CEA-2 cells in C57BL/6 mice [117] responses challenge (70% of mice)

Prolongation of survival time (70% of mice), CpG-ODN plus Tat-CEA fusion protein MC38-CEA-2 cells in C57BL/6 mice CTLs, IgG [118] reduction of tumor volume (100% of mice)

Modified CEA691 HLA-A2Kb transgenic mice CTLs NA [121]

CEA+/HS-Exo HLA-A2.1/Kb transgenic mice CTLs NA [127]

270 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al.

(Table 1) Contd….

Type of Vaccine Mouse Model Effector Anti-Tumoral Effect Reference

COMBINED VACCINATION

Prime DNA-protein boost vaccination

Prime (hCEA/DNA)-boost (rCEA/protein) CTLs, CD4+T cells, BALB/c mice Protective immunity [131] pKCEA66 plasmid in combination with antibodies, IFN- rCEA protein

Prime (hCEA /DNA)-boost (rCEA/protein) Protective immunity BALB/c mice IgG [132] pKCEA66 plasmid in combination with (50%) rCEA protein

Prime (hCEA/DNA)-boost (hCEA/DNA)

Protective immunity BALB/c mice IgG, CTLs, IFN- [132] pKCEA66 plasmid in combination with (100%) twtCEA DNA encoding the wild type CEA fused a tetanus T helper epitope

Prime DNA-virus boost vaccination

Protective immunity (80%) ; prolonged hCEA/DNA (pCMV-CEA) plus ADHSP72 TrampC2-CEA cells in C57BL/6 mice IgG, CTLs survival (10 days more [133] than in the control group)

Prime (pV1J/CEA-DOM)-boost (Ad/CEA– Antibodies, CTLs, Suppression of tumor MC38-CEA cells in CEA tg mice [134] DOM) IFN- growth (87%)

Prolongation of tumor- Prime (pV1J/CEA-LTB)-boost (Ad/CEA- Antibodies, CTLs, MC38-CEA cells in CEA tg mice free survival time (80% [135] LTB) IFN- of mice)

Prime (rhCEA/DNA)-boost (AdV-CEA) Antibodies, CTLs, Suppression of tumor MC38-CEA cells in C57BL/6 mice [136] pV1J-rhCEA in combination with Ad5- IFN- growth (57%) rhCEA

Prime (hCEA/DNA)-boost (AdV-CEA) DMH-induced colon lesions in CEA tg Reduction of tumor mice and APC1638N/CEA hybrid Antibodies, CTLs [137] multiplicity and volume pV1J/CEA-LTBopt in combination with transgenic mice Ad/CEA-LTBopt

Prime virus-protein boost vaccination

Antibodies, T-cell Protection from tumor Prime (rV-CEA)-boost (bV-CEA) MC38-CEA-2 cells in C57BL/6 mice [143] responses challenge (70% of mice)

*CEA-Positive Lewis lung carcinoma fected with CEA and the Lewis lung carcinoma cell line ex- observed [28]. To improve the protective effect of the oral pressing human CEA [27, 28]. Indeed, 4 of 8 mice orally vaccines, the CEA-based DNA vaccines have also been vaccinated with the pCD40LT-CEA plasmid, which encodes combined with a fusion protein composed of human IL-2 and the fusion protein consisting of human CEA and mouse the carboxyl terminus of the Fc fragment of human IgG1, as CD40 ligand, efficiently rejected the tumor cell challenge boost [27-30]. [27]. Similarly, in mice orally vaccinated with the pW-CEA The oral DNA vaccine encoding CEA and the CD40 plasmid and then challenged with Lewis lung carcinoma ligand and the methods of using them were patented by cells positive for CEA and epithelial cell adhesion molecule Xiang et al. The DNA vaccine was administered orally to Ep-CAM/KSA, a reduction of tumor growth to 25% was CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 271 mice and was effective against the growth of CEA- lenge with the MC38-CEA-2 cells than mice immunized expressing cancer cells [31]. with CEA-DNA alone [37]. Similarly, novel double transgenic mice (CEA-A2Kb), A novel adjuvant, the oral toll-like receptor 7 agonist which are peripherally tolerant to CEA, have been used to SM360320, was used in CEA-transgenic mice (CEA.Tg) to investigate the efficacy of an oral CEA-DNA-based vaccine. improve the efficacy of in vivo electroporation of the CEA- These mice were orally immunized with the CEA pHI- DNA plasmid. Treatment with a combination of DNA-EP 691DNA minigene vaccine encoding for CEA691 peptide and SM360320 led to a significant anti-tumor effect, which (IMIGVLVGV), which was able to break the peripheral T was associated with the anti-CEA antibody isotype switch cell tolerance against CEA. After immunization, a CEA- from IgG1 to IgG2a [38]. specific antitumor CTL response, which conferred protective immunity against a challenge with CEA-A2Kb-transduced Anti-idiotype Antibody-based Vaccines MC-38 colon carcinoma cells has been observed, thus confirming the potency of oral DNA vaccination as a candidate The use of anti-idiotype (anti-Id) antibodies to induce for human vaccine development [32]. TAA-specific humoral responses has been explored. Several monoclonal anti-Id antibodies that mimic distinct epitopes of The combination of naked DNA with immune-stimulants human CEA have been developed. Bhattacharya-Chatterjee is another potential strategy for enhancing the efficiency of and co-workers developed a monoclonal anti-Id antibody, DNA vaccination. Song et al. demonstrated that co- named 3H1 (Ab2), that mimics a specific epitope of human vaccination with a human CEA-DNA plasmid and an IL-12 CEA [39]. This anti-Id antibody was used as a tumor vaccine plasmid resulted in a stronger protective anti-tumoral effect against CEA in several murine cancer models. Preliminary than vaccination with CEA DNA alone. C57BL/6 mice were studies performed with C57BL/6 mice showed that immuni- transplanted with CEA-positive Lewis lung carcinoma zation with the 3H1 antibody induced high titers of anti-CEA (CEA/LLC) cells and then immunized with a plasmid encod- antibodies and cellular responses that protected mice against ing human CEA and a plasmid encoding IL-12. The co- challenge with lethal doses of MC38-CEA cells, resulting in injection of these two plasmids was able to enhance both the only minimal tumor growth [40]. In addition, CpG- anti-CEA humoral response and the cell-mediated immune oligodeoxynucleotides (CpG-ODN) (1826) enhanced the response and efficiently counteracted mouse tumor growth. potency of the 3H1 antibody-based vaccine in the same mur- Indeed, 80% of these mice remained tumor-free after vacci- ine model [41]. nation. The authors concluded that the production of CD4+ and CD8+ T cells could play an important role in counteract- Moreover, the adjuvant effect of CpG-ODN (1826) and ing tumor growth [33]. QS-21 in the anti-idiotype (anti-Id) antibody vaccine was also evaluated in transgenic mice expressing human CEA Similarly, the administration of a plasmid encoding [C57BL/6J-TgN (CEAGe) 18FJP]. The co-administration of membrane-bound IL-4 or IL-12 increased the effects of 3H1 with CpG-ODN (1826) resulted in a better induction of CEA-DNA vaccination. All mice vaccinated using the com- anti-tumor immune responses, especially CTL responses, bination of the human CEA plasmid with the IL-4 or IL-12 compared to the co-administration of 3H1 with QS-21. In plasmid showed strong anti-CEA immunity and rejected addition, mice immunized with 3H1 plus CpG-ODN (1826) tumor growth upon challenge with CEA/LLC cells [34]. showed a longer survival compared to 3H1-QS21-vaccinated Moreover, it has been demonstrated that co-delivery of a mice. These results suggested that CpG-ODN (1826) is an plasmid encoding human CEA and a plasmid encoding the optimal adjuvant to enhance the potency of the 3H1 vaccine soluble B7.1/IgG Fc fusion protein induced potent antitumor [42]. immunity and an efficient tumor growth inhibition in The amino acid sequence of the variable regions of the C57BL/6 mice transplanted with CEA/LLC cells. Although monoclonal anti-Id 3H1 antibody and a polynucleotide se- the B7.1/IgG Fc fusion protein did not elicit any immune quence encoding these variable regions are provided in the response alone, it was an effective co-stimulatory agent that US5977315 patent. Moreover, this patent provides the phar- enhanced the ability of DNA vaccination to induce cell- maceutical compositions and vaccines comprising an effec- mediated immunity, especially through the activation of tive amount of 3H1 in combination with an adjuvant that + CD8 naïve T cells [35]. A plasmid encoding a mutant B7- enhances the presentation of 3H1 and the immune response 1/Ig fusion protein has also been developed, which amelio- against 3H1 [43]. rates the DNA vaccination against CEA and tumor rejection by binding to CTLA-4 on T cells. When this plasmid is used In the US7090842 patent, the same inventors also dis- as a vaccine, the CTLA-4/B7-1 binding during T cell/T cell closed the design of diagnostic kits and other methods of interaction is blocked. This blockage leads to the inhibition using 3H1 polynucleotides, including methods of treating of negative regulatory signals, thus improving anti-tumor CEA-associated tumors with the 3H1 antibody. In this pat- immunity. Mice vaccinated with the plasmid encoding hu- ent, polynucleotides encoding polypeptides with the immu- man CEA and mutant B7-1/Ig showed a reduced tumor inci- nological activity of the monoclonal anti-Id antibody 3H1 dence of 60% [36]. Similarly, a fusion DNA plasmid encod- are described. These polynucleotides encode sequences of ing human CEA and mGM-CSF was used as a DNA vaccine both the variable light and heavy chain of the 3H1 antibody. to immunize C57BL/6 mice. Mice immunized with this Accordingly, the 3H1 polynucleotides can be inserted in live plasmid showed both CEA-specific anti-tumor cellular and or attenuated viruses (vaccinia virus) or viral vectors, such as humoral responses and were better protected against a chal- adenovirus, adeno-associated viruses (AAV) and SV40, which are used for vaccine formulations [44]. 272 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al.

Furthermore, therapeutic uses of the 3H1 antibody for against tumors by priming T-cell responses to target TAAs. treating CEA-associated tumors (gastrointestinal origin, co- The absence of antigen presentation by tumor cells is mainly lorectal and lung) in high-risk individuals are described in due to the inhibition of DCs maturation, migration and cross- two other US patents. The US7300651 patent provides presentation functions, which leads to an inactivation of T- methods for the administration of the anti-idiotype 3H1 anti- cell responses that confers the ability of tumor cells to escape body to cancer patients. It has been suggested that the 3H1 immunosurveillance [53]. The use of DCs loaded with CEA antibody can be administered in combination with an adju- enhances presentation of the antigen to the host immune sys- vant (aluminum hydroxide) or with other pharmaceutical tem. In this regard, several strategies involving DCs-based drugs, such as 5-fluorouracil and levamisole hydrochloride vaccines have been employed. or leucovorin calcium [45]. One strategy includes the use of DCs pulsed with protein In another patent, methods and compositions are de- or synthetic peptides derived from CEA. Wu et al. pulsed scribed for treating HMFG- and CEA-associated tumors DCs with a CEA-Hsp70-like protein 1 (CEA-Hsp70L1) fu- through the combined administration of the anti-Id antibody sion protein and showed that the fusion with the Hsp70L1 11D10 for HMFG and the anti-Id antibody 3H1 for CEA protein promoted DCs maturation and also enhanced the [46]. production of cytokines and chemokines. In this regard, b Similarly, Losman et al. developed a rat monoclonal anti- HLA-A2.1/K mice immunized with DCs pulsed by CEA- Hsp70L1 showed a more efficient induction of CEA-specific Id antibody (rW12) specific for the murine anti-CEA mono- CTLs than mice immunized with DCs pulsed with CEA clonal antibody MN-14. The authors demonstrated that im- alone. Moreover, the adoptive transfer of splenocytes derived munization with rW12 was able to induce an anti-CEA hu- from these immunized mice inhibited tumor growth in nude moral response in mice and rabbits [47]. mice bearing CEA+/HLA-A2.1+ human colon carcinoma, A chimeric form and a humanized form of rW12 have confirming the potency of the use of the Hsp70L1 fusion been described in a US patent application. In particular, two protein as an adjuvant in DCs-based cancer vaccination [54]. isolated polynucleotides that encode the heavy and the light The use of DCs pulsed with an Escherichia coli-derived chain variable regions of the chimeric or humanized forms of recombinant Hiv-Tat/human CEA fusion protein was inves- rat W12 are provided. In addition, a method of administering tigated in a mouse tumor model (B6 mice inoculated with the chimeric or humanized form of rW12 conjugated to a soluble immunogenic carrier protein, optionally in combina- MC38-CEA2 cells). Mice immunized with Tat-CEA-pulsed DCs exhibited a stronger specific anti-CEA CTL response, tion with an adjuvant, has been described [48]. which conferred better protection against tumor growth Durrant et al. produced a mouse anti-Id antibody (708) compared with mice immunized with CEA-pulsed DCs or that mimics CEA. However, this anti-Id antibody did not DCs alone [55]. demonstrate good immunogenicity [49]. The US200502- 22392 patent application provided modified versions, which A fusion protein comprising a nucleotide sequence encoding Hiv-Tat protein, one of the well-known PTDs (pro- improved the immunogenicity of the mouse antibody 708. tein transduction domain), and a nucleotide sequence encod- Sequence tracts derived from CEA and CEA cancer-specific ing human CEA was described in the US7364743 patent. MHC epitopes were introduced into the variable regions of PTDs are able to transduce external proteins into the cyto- the 708 antibody. Accordingly, the modified 708 antibody plasm to improve the MHC class I antigen presentation and was able to induce a strong immune response to CEA [50]. to enhance CTL responses. In addition, this invention de- The efficacy of a fusion protein composed of a single- scribes a cancer vaccine and a pharmaceutical composition chain variable fragment of an anti-Id antibody mimicking an comprising the Tat-CEA fusion protein for treating tumors epitope specific for human CEA (scFv6G6.C4) and mGM- and the method for inducing CEA-specific antitumor immu- CSF in overcoming peripheral tolerance in CEA-transgenic nity using mammalian DCs pulsed with Tat-CEA [56]. mice has also been investigated. C57BL/6 mice transgenic for the human CEA that were immunized with this fusion Similarly, Kim et al. pulsed DCs with an mRNA encoding a fusion protein composed of TAT-PTD, calreticulin protein showed stronger anti-CEA antibody responses to (CRT) and a human truncated CEA (CRT-TAT-CEA) to both scFv6G6.C4 and CEA than mice immunized with enhance CEA-specific immune responses. C57BL/6 mice scFv6G6.C4 alone. The authors concluded that the use of were first inoculated with MC38/CEA2 cell lines and then immunostimulatory molecules, such as GM-CSF, potentiated vaccinated with DC/CRT-TAT-CEA. After immunization, the capacity of anti-Id antibody vaccines to break peripheral + + tolerance against TAAs [51]. enhanced CEA-specific CD4 and CD8 T cell responses were observed, resulting in the inhibition of tumor growth Similarly, Pignatari and co-workers developed an anti-Id and prolonged survival [57]. scFv isolated from an anti-Id monoclonal antibody (6.C4) Moreover, a European patent describes a vaccine based that mimicked CEA. The authors demonstrated that this scFv on DCs loaded both with the cDNA encoding for CEA and was able to mimic CEA and stimulate a humoral response against CEA in BALB/c mice [52]. with the CEA protein. The double loading of DCs ensures the activation of CD8+ T cells and B-cells [58]. Dendritic Cell-based Vaccines DCs were also pulsed with the monoclonal anti-idiotype antibody 3H1. Transgenic CEA mice (CEA.Tg) immunized Professional antigen-presenting cells (APCs), such as with 3H1-pulsed DCs showed high titers of anti-CEA- DCs, play an important role in inducing immune responses specific IgG antibody, an improved activation of CD4+ T CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 273 cells and an enhanced induction of CEA-specific CTL re- Another strategy of vaccination that employs DCs trans- sponses. The generation of both humoral and cellular anti- duced with adenovirus carrying the human CEA, GM-CSF CEA specific responses conferred CEA.Tg mice protection and interleukin 12 (IL-12) genes has also been investigated. against a challenge with CEA-transfected C15 tumor cells. This DCs-based vaccine led to the generation of potent im- Moreover, the authors suggested that 3H1-DCs vaccination mune responses against syngeneic MC38-CEA tumor cells in was better than CEA-DCs vaccination at inducing antitumor CEA-transgenic mice [C57BL/6J-TgN (CEA Ge) 18FJP] immune responses [59, 60]. These authors also investigated and conferred to these mice the capacity to eradicate the tu- the efficacy of a combined vaccination strategy using DCs mor. This antitumor immune response involves the activa- pulsed with CTL epitopes from CEA and the monoclonal tion of CEA-specific CD4+ T cells, which play a crucial role antibody 3H1 in improving T-helper and CTL responses in for the activation of NK cells and CD8+ T cells, which are in C57BL/6J-CEA-A2Kb mice bearing MC-38-CEA-A2Kb turn necessary for tumor regression [66]. colon carcinoma cells. The combined vaccine conferred to Moreover, because the expression of different TAAs, mice a significant increase in survival and the ability to such as survivin (SVV), has been observed in human colo- eradicate tumors compared with vaccination with peptide rectal cancer, Kim et al. investigated whether adenovirally pulsed DCs alone [61]. transduced DCs that simultaneously express CEA and SVV In addition, these authors investigated the effects of CpG- were able to enhance the antitumor immunity against CEA in oligodeoxynucleotides (CpG-ODN) as adjuvant molecules to C57BL/6 mice injected with MC38/CEA2 tumor cells. To potentiate the antitumor activity of DCs pulsed with the CTL improve the efficacy of DCs that have been co-transduced peptides of CEA plus the monoclonal anti-Id antibody 3H1 with adenovirus vectors encoding CEA and SVV, an adaptor in the murine colon carcinoma model C57BL/6J-CEA- fusion protein, which combines coxsackie and adenovirus A2Kb. Immunized mice showed a significant increase in the receptor (CAR) with the TAT-protein transduction domain inhibition of tumor growth and long-term survival when (TAT), named CAR-TAT, has been employed. In this study, CpG-ODN was administered in combination with these DCs the authors demonstrated that DCs expressing CEA and SVV compared with mice immunized with DCs pulsed with CTL in the presence of CAR-TAT led to a more efficient induc- peptides of CEA plus the monoclonal anti-Id antibody 3H1. tion of both CEA- and SVV-specific T-cell responses and These results suggested that CpG-ODN exerts a potent adju- also led to increased cytotoxic activity against the vant activity, especially through the induction of CEA- MC38/CEA2 cell line compared with DCs transduced with specific CD8+ T cells, the activation of NK cells and the the adenovirus expressing CEA and SVV alone and com- promotion of the long-term survival of CD8+ T effector and pared with adenovirally transduced DCs expressing both memory T cells [62]. CEA and SVV in the absence of CAR-TAT. These data confirmed that a DCs-based vaccine targeting multiple TAAs The potency of vaccination with DCs pulsed with CTL was more efficient in eliciting antitumor responses than a peptides from CEA plus 3H1 was also enhanced through the + DCs vaccine targeting a single TAA [67]. These authors also depletion of CD25 T cells and CTLA-4 blockade. The best protection against tumor growth was achieved when demonstrated that the combined therapy using a DCs-based vaccine expressing human CEA and a chemotherapy regi- C57BL/6J-CEA-A2Kb mice were immunized with DCs men with irinotecan plus infusional 5-fluorouracil and leuco- pulsed with the CTL peptides of CEA plus 3H1 in combina- vorin (FOLFIRI) had enhanced antitumor properties because tion with anti-CD25 and anti-CTLA-4 antibodies, suggesting it was able to inhibit the immunosuppressive tumor envi- that the simultaneous blockade of T-cell regulatory pathways ronments in a colorectal cancer murine model [68]. could play a role in enhancing the therapeutic efficacy of DC-based vaccines in the treatment of CEA+ colon cancer Recently, pharmaceutical compositions containing an [63]. adenovirus that encodes CEA, an adaptor fusion protein comprising a CAR, and the human CD40 ligand were pro- Another strategy to improve the DCs-based vaccination posed in a US patent application. The inventors observed is the transduction of DCs by recombinant adenovirus that the human CD40 ligand and the CAR ectodomain are (rAdV)-expressing TAAs. Oh and co-workers demonstrated that vaccination with DCs transduced with rAdV encoding useful for promoting adenoviral transduction and the maturation of DCs and that they are also useful for promoting im- the CEA gene (AdV-CEA) was more efficient than vaccina- mune response to CEA [69]. tion with CEA peptide-pulsed DCs in inducing antitumor immune responses in a murine tumor model (C57BL/6 mice). Vaccination with AdV-CEA/DCs promoted an im- Recombinant Vector-based Vaccines provement in the proliferation of antigen-specific T cells and One strategy to overcome the tolerance to CEA and to the number of IFN- secreting T-cells, and it also induced induce protective immunity is the use of recombinant viral, potent anti-CEA-specific CTL responses in C57BL/6 mice. bacterial or yeast vectors expressing human CEA. In this Moreover, generation of these immune responses induced regard, it has been observed that the TAAs delivered by the ability to counteract tumor growth and conferred the vac- these vectors were more immunogenic than TAAs adminis- cinated mice with complete protection after a challenge with tered alone or in combination with adjuvants [70]. MC38/CEA2 tumor cells [64]. In this regard, the US8012468 patent describes an antitumor vaccine and pharmaceutical a) CEA-viral Vectors composition for treating tumors expressing CEA that com- The first evidence of the efficacy of using viral vectors as prises DCs transduced with rAdV-CEA or CEA-specific vehicles for TAAs was reported by Kaufmann et al., who CTLs generated by DCs transduced with rAdV-CEA [65]. immunized mice with a recombinant vaccinia virus that ex- 274 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al. pressed human CEA (rV-CEA) and observed that the ad- in a more efficient manner. In addition, Tg mice were trans- ministration of this vector elicited a humoral immune re- planted with MC-38-CEA-2 cells and, after 14 days, were sponse against CEA. In addition, mice bearing a syngeneic treated by immunization with rV-CEA/TRICOM vectors colon adenocarcinoma expressing human CEA were able to alone or in combination with GM-CSF and IL-2. The authors reduce tumor growth after the administration of rV-CEA observed that mice immunized with rV-CEA/TRICOM vec- [10]. tors in combination with GM-CSF and IL-2 showed a significant increase in survival compared with mice that re- One strategy to enhance the effects of rV-CEA is the ceived the rV-CEA/TRICOM vectors alone (83% vs. 14%) combined administration of rV-CEA and co-stimulatory [76]. molecules. A first example comes from the pioneering work of Hodge et al., who reported on the efficacy of the com- Similar results were obtained by Grosembach et al., who bined administration of rV-CEA and recombinant vaccinia extensively studied the differences in T-cell responses upon virus expressing the co-stimulatory protein B7-1 (rV-B7). In the use of different protocols that employ rV- this context, immunization of C57BL/6 mice with rV-CEA CEA/TRICOM, a recombinant avipox (fowlpox, rF) vector and rV-B7 at a 3:1 ratio resulted in significant lymphoprolif- expressing CEA/TRICOM alone or in combination with the eration in response to CEA, together with CEA-restricted T- rF-GM-CSF vector (TRICOM/diversified prime and cell responses. Moreover, the vaccination protected mice boost/GM-CSF). These authors demonstrated that the syner- from tumor challenge with MC-38-CEA-2 cells and induced gistic vaccine strategy was a useful approach to amplify the capacity to reject tumor establishment in the vaccinated CEA-specific immune responses and antitumor effects [77]. mice. In this regard, 80% of mice immunized with rV-CEA Moreover, vaccination with rV-CEA/TRICOM vectors alone and rV-B7 at a 3:1 ratio efficiently counteracted tumor or in combination with GM-CSF was able to induce a potent growth [71]. immune response to CEA without inducing autoimmunity or The same authors also patented a composition containing other pathologies in different mouse models bearing transgenic CEA expression [78, 79]. a combination of rV-CEA and rV-B7-1 or rV-B7-2 for up- regulating cell-mediated immunity as well as humoral im- The efficacy of a subcutaneous priming vaccination with munity against CEA in mice bearing CEA+-tumors [72, 73]. rV-CEA/TRICOM plus rF-GM-CSF followed by intratumoral boosting vaccination with rF-CEA/TRICOM plus rF- McLaughlin et al. demonstrated that IL-2 administration enhanced the therapeutic effects of rV-CEA in C57BL/6 IL-2 and rF-GM-CSF was confirmed by Kudo-Saito et al., who observed a complete tumor regression in 80% of (B6; H-2b) mice through an improvement of CEA-specific C57BL/6 mice Tg for human CEA transplanted with MC-38- T-cell responses. Moreover, it has been observed that immu- CEA-2 cells [80]. nization with rV-CEA plus interleukin 2 (IL-12) 10 days after a challenge with MC-38-CEA-2 cells could induce a In addition, Kudo-Saito et al. also demonstrated a sig- regression of tumors in 60-70 % of mice. Immunization also nificant therapeutic effect resulting from the simultaneous resulted in prolonged survival and induced a significant de- administration of rV-CEA/TRICOM with an rV vector ex- lay in the growth of MC-38-CEA-2 tumors [74]. In light of pressing 4-1BBL (CD137), a TNF family member strongly these promising results, a pharmaceutical composition con- involved in both the activation and the long-term mainte- taining rV-CEA in association with a biological response nance of primed T cells. The combination of rV4-1BBL and modifier, such as IL-2, IL-6, interferon or cyclophos- rV-CEA/TRICOM resulted in a significant activation of phamide, intended to elicit a humoral and/or cell mediate CEA-specific CD4+ and CD8+ T cells and a significant de- response in vivo directed against CEA or cells expressing crease in tumor growth in CEA-Tg mice implanted subcuta- CEA has been provided by Schlom et al. in the US5698530 neously with MC38-CEA+ tumors [81]. In addition, the com- patent [75]. bined dietary administration of the COX-2 inhibitor Cele- coxib (at a dose of 1000ppm) and the subcutaneous injection Another authoritative example of the promising use of of rV-CEA-TRICOM resulted in a highly significant in- combined vaccination with vaccinia virus plus costimulatory molecules is represented by the work of Aarts et al., which crease in the survival of mice bearing multiple CEA+ intestinal neoplasms [82]. reported the efficacy of a vaccination protocol consisting of the combined administration of several vaccinia viruses con- A similar strategy to increase the effectiveness of rV- taining the human CEA transgene and three T-cell costimu- CEA/TRICOM and rF-CEA/TRICOM has also been pro- latory molecule transgenes [B7-1, ICAM-1, and LFA-3, posed by Chakraborty et al., who demonstrated that con- called recombinant vaccinia (rV)-CEA/TRICOM], with each comitant “negative signal blockade” through the anti-CTLA- transgene under the control of individual poxvirus promot- 4 mAb 9H10 was highly effective in generating CEA- ers. The results from this study demonstrated that continued specific CD8+ T cells compared with rV-CEA-TRICOM boosting with this vaccine is required to maintain a CEA- alone and was also effective in inducing a 30-fold higher specific T-cell response in C57BL/6 mice transgenic (Tg) for cytolytic effect in comparison to standard rV-CEA-TRICOM human CEA and that the combined administration of rV- plus rF-GM-CSF vaccination. Accordingly, the authors CEA/TRICOM with local GM-CSF together with a low dose demonstrated an antitumor synergy between the CTLA-4 of systemic IL-2 was essential to potentiate the effects of rV- blockade and vaccination with rV-CEA/TRICOM-rF-CEA/ CEA/TRICOM vectors. Mice immunized with rV-CEA/ TRICOM in CEA-Tg mice injected with MC-38-CEA-2. A TRICOM in combination with GM-CSF and IL-2 showed an total of 20% of mice that received mAb 9H10 in association enhanced CEA-specific T-cell proliferation and IFN- pro- with vaccine therapy remained tumor-free for a long period duction, which allowed mice to counteract tumor progression of time [83]. CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 275

The same authors also proposed an alternative strategy to responses and potent humoral responses, but not by CTL- increase the efficacy of rV-CEA/TRICOM and rF-CEA/ specific response. CEA-specific CTL responses were ob- TRICOM. They administered an yttrium-90 (Y-90)-labeled served only in the presence of concomitant CEA-DNA vac- anti-CEA mAb (derived from the anti-CEA murine mAb cine administration, suggesting the generation of different T- COL-1) together with the vaccines. This combinatory strat- cell repertoires in response to different vaccination strategies egy was far more effective in inducing antitumor responses, [91]. almost doubling the half-life of survival in mice bearing In a recent US patent, Schlom et al. described a pharma- CEA+ tumors treated with vaccines alone [84]. ceutical composition containing a recombinant vector encod- Similarly, Farsaci et al. recently reported that the admini- ing human CEA and recombinant vectors encoding and ex- stration of the small molecule BCL-2 inhibitor GX15-070 is pressing B7-1, ICAM-1, and LFA-3 (CEA/TRICOM vec- strongly effective in augmenting the effects of the vac- tors). The recombinant vectors of this invention included cinia/fowlpox CEA-TRICOM vaccine, in terms of both regu- bacterial vectors, nucleic acid-based vectors and viral vec- latory T cell (Tregs) depletion and tumor reduction in CEA- tors, such as poxvirus vectors. Poxvirus could be selected Tg mice transplanted with LL2-CEA tumor cells [85]. These from vaccinia-Copenhagen, vaccinia-Wyeth strain, vaccinia- observations are also coherent with reports from Hance et MVA strain, fowlpox, canarypox, ALVAC, and swinepox. al., who recently demonstrated the efficacy of IFN- ad- In cancer treatment, the virus-based vectors can be adminis- ministration together with the CEA-TRICOM vaccine in tered through the exposure of mammalian tumor cells to the inducing NK- and CD8+-dependent tumor lysis as well as recombinant virus ex vivo or through injection of the recom- antigen presentation, thus resulting in tumor growth inhibi- binant virus into the affected host by intravenous, intrader- tion and improved survival in mice bearing CEA+ tumors mal or intramuscular administration [92]. In addition, [86]. Schlom and colleagues also described a pharmaceutical The need for the attenuation of potential adverse reac- composition containing recombinant poxvirus vectors encoding for CEA administered by intradermal scarification or by tions by viral vectors, including those based on replication- intramuscular, subcutaneous and intravenous routes with a competent poxviruses, is the founding idea for the use of biological adjuvant such as IL-2, IL-6, IL-12, IL-15, B7.1 or modified vaccinia Ankara (MVA), a replication-defective B7.2. These molecules potentiate the effect of vaccination form of vaccinia virus. Convincing results in this field have with recombinant poxviruses. These adjuvants can be admin- been provided by Hodge et al., who developed and tested an MVA bearing both the CEA and the co-stimulatory triad istered as a whole protein or an immunogenic peptide fraction of the same protein or as DNA encoding the protein or transgenes (called rMVA-CEA/TRICOM). This vector, in + + the peptide fraction [93]. Similarly, the use of a combination fact, provided potent CEA-specific CD4 and CD8 T-cell of recombinant poxviruses, such as NYVAC or ALVAC, responses and also yielded a significant therapeutic antitu- expressing the human CEA gene and the immunostimulatory mor response, although only two administrations in vaccinia- molecules IL-2, IFN-, IL-4, GM-CSF, IL-12, and B7 for the naive mice and one in vaccinia-immune mice were possible before the induction of anti-vector immune responses. To treatment of cancer has been claimed in the US6780407 patent [94]. overcome this problem, the same authors proposed that priming with rMVA-CEA/TRICOM a week after by an rF- Adenoviral (Ad) vectors are also used as carriers to de- CEA/TRICOM boost at the same injection site generates liver the human CEA protein into the host to break the toler- superior tumor-specific immunity and antitumor activity [87, ance to CEA, but their application in immunotherapy proto- 88]. cols remains controversial because of the pre-existent immunity against adenovirus present in several animal models and Intriguingly, the results from Hodge et al. demonstrated in humans. In this regard, because anti-adenovirus immunity that the CEA-expressing canarypox virus (ALVAC-CEA), is becoming a critical issue affecting the efficacy of repeated an avipoxvirus-based vector, was safer than the standard Ad vector vaccinations, Gabitzsch et al. have recently devel- poxvirus vectors in inducing humoral and cytolytic T-cell oped a novel Ad5 vector bearing deletions of the viral DNA responses in C57BL6 mice. Moreover, the authors observed that mice that were immunized with rV-CEA and then polymerase and the pre-terminal protein region (Ad5 (E1, E2b-). The CEA gene was inserted into this vector, demon- boosted with ALVAC-CEA showed a significant improve- strating that the Ad5 (E1-, E2b-)-CEA vector could induce ment of CEA-specific T-cell responses, which conferred cell-mediated immunity and tumor growth inhibition in Ad5 good protection against tumor growth after a challenge with pre-immunized mice bearing CEA-expressing tumors. Fur- MC-38-CEA-2 in these mice [89]. thermore, the authors observed that the pre-existing Ad5 Kass et al. reported that the concomitant administration immunity did not affect the efficacy of this vector and con- of ALVAC-CEA and the avipoxvirus-expressing mGM-CSF cluded that the novel Ad5 (E1-, E2b-)-CEA vector can be (ALVAC-GM-CSF) significantly enhanced CEA-specific used for multiple re-immunizations without causing signifi- humoral and cell-mediated immunity in CEA.Tg mice. Fur- cant side effects [95]. thermore, vaccinated mice were able to reject the tumor chal- Methods and compositions for producing these recombi- lenge, and five mice became tumor free after a challenge with MC38-CEA-2 cells [90]. nant adenoviral vectors that allow multiple vaccinations with the same adenovirus vector and generate immune responses The efficacy of the avipoxvirus-based vector was con- while avoiding side effects due to the preexisting immunity firmed by Bos et al., who demonstrated that ALVAC-CEA to adenovirus were disclosed by the same authors in the induced a protective CEA-specific immunity by CD4+ T cell US20100183673 application patent. To administer the vac- 276 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al. cine, recombinant adenoviral vectors were combined with an also resulted in increased CEA levels in the peripheral blood appropriate buffer, a physiologically acceptable carrier and as a sign of viral replication [102]. excipients, to be administered intravenously, intraperito- Facciabene et al. reported that the in vivo delivery of ba- neally, intramuscularly and also in the form of pills [96]. culovirus vectors expressing the E2 glycoprotein of hepatitis Similarly, in the US20110059135 patent application, C virus or CEA (Bac-G-E2 and Bac-CEA, respectively) can Kovesdi et al. proposed modified adenoviral vectors opti- effectively elicit an antitumor response by inducing specific mized for CEA delivery. These vectors were constructed to + CD8 T cell- and NK-dependent responses [103]. incorporate an epitope or an antigen into a capsid protein that could be administered to a mammal several times to any Baculovirus-based vaccines, used to elicit both humoral suitable tissue or delivered systemically in association with a and cellular immune responses against a specific antigen physiologically acceptable carrier [97]. expressed by the baculovirus in an animal, are described in the US20100285056 patent application. These vaccines can Another strategy to overcome anti-adenovirus immunity be formulated in a unit dose form and in a multidose form, has been proposed by Selvarangan et al., who developed a and the pharmaceutical compositions include a solution, a CEA-expressing recombinant adeno-associated virus 2 suspension or an emulsion in oil or aqueous medium, an (CEA-rAAV) vector and tested it for its ability to induce a elixir, a powder, a granule, and a tablet and may also com- protective antigen-restricted antitumor response. Interest- prise of a dispersion agent and a stabilizer [104]. ingly, the authors observed that although CEA expression in mice infected with the vector was strong, only a humoral Stabilizing formulations for recombinant viruses that can anti-transgene response could be detected, and both cellular be used as vaccines were patented by Sandrine. The formula- immunity and antitumor effects were negligible. Interest- tions comprise a sugar (i.e., sucrose or sorbitol), a preserva- ingly, however, if a bacterial DNA plasmid containing meth- tive, a dispersing agent (i.e., dextrane, PEG), a thermal sta- ylated motifs (pNGVL3) was injected three weeks later than bility agent (i.e., urea), a buffer (i.e., PBS, Hepes, borate, the viral vector, a significant in situ recruitment of DCs to- MOPS) and one or more distinct types of amino acids. These gether with a CEA-restricted T-helper-1 response could be formulations ameliorate the preservation and the stability of observed. C57BL/6 mice immunized with the combination recombinant viruses, allowing them to be maintained for a of viral and bacterial vectors were then challenged with long period of time without influencing their biological ac- MC38-CEA-2 cells. These mice showed a prolonged sur- tivity [105]. vival time and a marked increase in the percentage of tumor- b) CEA-bacterial Vectors free survival when compared with mice vaccinated with CEA-rAAV alone [98]. Bacterial vectors were used as well as viral vectors to deliver TAAs into a host because of their capacity to activate The use of other viral delivery systems was also investi- a proinflammatory response that is useful to break the toler- gated. Ansardi et al., developed a recombinant CEA- ance to a specific TAA. Although the use of bacterial vectors poliovirus replicon in which they demonstrated that cells is a promising approach for developing a CEA-based vac- infected with encapsidated replicons containing the CEA- cine, their application is still rarely used in animal models. poliovirus genome were able to express the CEA protein and Examples of bacterial vectors used in immunotherapy are that these encapsidated replicons were also capable of stimu- Escherichia coli, Salmonella, Shigella, and Listeria monocy- lating a humoral anti-CEA response when administered to togenes [15]. mice [99]. The US7700344 patent provides compositions, methods Similarly, Cusi et al. demonstrated that intranasal deliv- and kits for enhancing the immunogenicity of a TAA, such ery of immune-reconstituted influenza virosomes (IRIV), as CEA, through fusion to an ActA protein. This invention assembled with the CD40L gene (CD40L/IRIV), could im- describes the antitumor activity of a recombinant Listeria prove the protective immunity and the CTL response against vaccine strain expressing the fusion protein ActA-antigen. In CEA induced by an IRIV vaccine assembled with the CEA this regard, when this vaccine was administered to an animal, gene (CEA/IRIV) [100]. destruction of the existing tumor and the induction of anti- In addition, Huang et al. recently reported the develop- gen-specific lymphocytes capable of infiltrating tumors were ment of a papillomavirus pseudovirus encoding a truncated observed [106]. form of CEA lacking the NH -terminal signal peptide (PV- 2 Vaccine compositions containing modified free-living CEA) as an oral vaccine. The results from this study demon- microbes, such as Bacillus anthracis or Lysteria monocyto- strated that PV-CEA was able to overcome the immunologic genes, and methods of using them for the loading, activation tolerance to CEA in CEA-transgenic mice and also showed and/or maturation of antigen-presenting cells are provided in potent antigen-restricted CTL responses and a delayed the US7927606 patent. These microbes were modified to growth rate of CEA+ tumors in the same mouse model, an include a TAA in their genome and are attenuated for prolif- effect which could be further amplified by the concomitant eration. Microbes were attenuated by psoralens, a group of administration of an equal vector encoding for IL-2 [101]. compounds that form irreversible cross-links in the genomes Similarly, Phuong et al. recently described the in vivo activ- of bacteria after illumination with ultraviolet A light (UVA). ity of a CEA-expressing recombinant measles virus (MV- The process of attenuation did not alter the expression of the CEA) vaccine derived from the Edmonston vaccine lineage. heterologous gene and the modified microbes were thus al- This type of vector was demonstrated to significantly delay lowed to deliver the antigen into a host without generating the growth of U87 glioblastomas in BALB/c nude mice and side effects linked to microbe proliferation. In addition, vac- CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 277 cine compositions could also contain adjuvants, T-cell and for the prevention and/or treatment of a variety of dis- costimulatory molecules or antibodies, such as anti-CTLA-4 eases, included tumors [112]. [107]. The US20120107347 patent application relates to the c) CEA-yeast Vectors concurrent use of two immunotherapeutic compositions, comprising a recombinant virus vaccine (rV/F-CEA/TRI- Recombinant yeast vectors represent a good approach for COM) and a yeast-based vaccine (yeast-CEA). Each vaccine the delivery of exogenous antigens in a host. Saccharomyces targets the same antigen and both vaccines have the capacity cerevisiae is considered a safe vector that could be used to to induce cell-mediated immune responses. Inventors dem- delivery TAAs. The promising features of this vector are its onstrated that the combined treatment determines a reduction known total lack of pathogenicity and the possibility of being of tumor growth, increases survival, delays onset of disease heat killed before administration. in CEA-Tg mice bearing LL2-CEA tumors [113]. Wansley et al. demonstrated that a CEA-expressing S. cerevisiae strain (CEA-yeast) was able to break the toler- Protein- and Peptide-based Vaccines ance to CEA and to produce specific T CD4+ and T CD8+ responses in CEA-Tg-vaccinated mice. In addition, multiple The use of proteins or peptides to induce anti-tumor im- administrations of this vector increased CEA-specific T-cell mune responses has been investigated, and CEA-protein- responses without causing side effects, thus allowing the based vaccines have been demonstrated to have good immu- vaccinated mice to efficiently counteract tumor growth and nogenicity and safety. to survive for a long period of time after the injection of A baculovirus vector was used to express recombinant MC38-CEA-2 cells [108]. CEA in insect cells that are capable of performing post- Further confirmation of CEA yeast vaccine efficacy translational modifications of recombinant proteins. Indeed, comes from the work of Bernstein et al., who demonstrated it was demonstrated that the biological and immunological the ability of a transgenic yeast vector expressing human activities of recombinant proteins expressed by insect cells CEA to stimulate CEA-specific T CD4+ and T CD8+ re- are similar to those of native proteins [114]. sponses in C57BL/6 (H-2b) mice [109]. Salgaller et al. described the development of eight bacu- Moreover, Boehm et al. have recently tested the efficacy lovirus clones expressing the human CEA protein for the of CEA/TRICOM and CEA-yeast vaccines alone or in com- first time. These clones contained multiple CEA epitopes, bination, demonstrating that distinct TCR repertoires and T- which resulted from the expression of different variants de- cell responses are induced by vaccination with these two rived by recombination within the repeated domains of CEA carriers. These results demonstrated that the concomitant [115]. Subsequently, epitope-mapping studies performed by injection of CEA/TRICOM and CEA-yeast vaccines was Bei et al. indicated that the baculovirus clone expressing the much more effective in inducing antitumor responses than full-length human CEA gene (bV-CEA) produced a recom- these vectors alone. In this regard, CEA-Tg mice challenged binant CEA protein that was recognized by a panel of 24 with LL2-CEA tumor cells and then vaccinated with the anti-CEA monoclonal antibodies and showed immunogenic combined CEA/TRICOM and CEA-yeast vaccines showed a properties. In this regard, bV-CEA was able to elicit a strong more significant reduction of pulmonary metastasis than humoral response and appeared to be a more potent immu- mice vaccinated with the CEA/TRICOM or CEA-yeast vac- nogen than native CEA in C57BL/6 mice [116]. cines alone [110]. The use of a cationic liposome formulation (DOTAP) as Disparities in T-cell subpopulations following chemo- an adjuvant was shown to potentiate CEA-specific immune therapy/CEA-yeast-based vaccination regimens have also responses in mice immunized with bV-CEA. The authors been very recently observed by Gameiro et al., who demon- demonstrated that when the recombinant CEA protein was strated that the combination of cisplatin and vinorelbine administered to mice in combination with DOTAP, animals positively modulates those components of the immune sys- were able to mount an immune response that protected them tem that are involved in a direct antitumor activity, and they from the transplant of MC38-CEA-2 cells [117]. also displayed a synergistic effect with CEA-yeast vaccina- Furthermore, C57BL/6 mice were also vaccinated with tion protocols [111]. the co-administration of CpG-ODN and the Tat-CEA fusion Compositions and methods for treating and/or preventing protein to stimulate innate and adaptive immune responses cancer in an animal through the use of a yeast-based vaccine against CEA. CpG-ODN exerted its adjuvant effects by pro- are disclosed in the US8153136 patent. To enhance the sta- moting the increase of IgG2a production and the CEA- bility of expression of the foreign antigen in the yeast vector specific CTL response. In this regard, mice inoculated with and/or prevent the post-translational modification of the an- MC38/CEA2 tumor cells had a therapeutic antitumor immu- tigen by the yeast, the heterologous antigen is fused at its nity that conferred to them the capacity of counteracting tu- amino-terminal end to a synthetic peptide or a portion of an mor growth and prolonged survival [118]. endogenous yeast protein. In addition, the yeast vector ex- Novel strategies for improving the efficacy of protein- pressing a TAA might also contain a yeast vector expressing based vaccines are described in the US20060275777 patent biological response modifiers, such as cytokines, lipidic de- application. Accordingly, this invention describes protein rivatives, hormones, and growth modulators. These composi- antigens (TAAs or viral proteins) covalently or noncova- tions can be used in prophylactic and/or therapeutic vaccines lently linked to the surface of lipid-based vesicles, such as liposomes or proteoliposomes. This invention describes 278 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al. compositions for parenteral administration, which comprise a application. The authors also suggested that the peptide- solution of the compositions dissolved or suspended in an based vaccine could be employed in the form of a ho- acceptable carrier, preferably an aqueous carrier. The inven- mopolymer comprising multiple copies of the same peptide tors also demonstrated that mice vaccinated with these com- or as a heteropolymer of various peptides mixed with an ad- positions showed a marked resistance to tumor growth and juvant, such as incomplete Freund’s adjuvant, aluminum progression and developed protective immunity after vacci- phosphate, or aluminum hydroxide [128]. nation [119]. Two US patent applications describe novel modified In the US8178653 patent, a vaccine based on a fusion CEA agonist or antagonist peptides, polypeptides and pro- protein which comprises CEA and a chain or a part of an teins containing a modified epitope. These patents describe MHC molecule is described. The administration of this mixtures and compositions of these agents alone or com- fusion protein to mice and humans, induces a strong acti- bined with one or more costimulatory molecules, such as vation of both CEA-specific CD4+ T and CD8+ T cells [120]. GM-CSF, IL-4, ICAM-1, LFA3, B7.1, B7.2, IFN, or TNF. One peptide described in this invention is a peptide agonist Peptide-based cancer vaccines are considered one of the or antagonist of the native CEA epitope, CAP-1. The agonist most specific strategies for polarizing the immune system peptide is able to elicit antigen-specific CTLs that inhibit the against a tumor that expresses a specific TAA. Unfortu- nately, this vaccine approach presents some drawbacks, such growth of or kill carcinoma cells expressing CEA or CEA epitopes, whereas the antagonist peptide serves to inhibit or as the ability to target only one or few epitopes, the poor prevent CEA-specific immune responses. The antagonist is immunogenicity of certain peptides, or the limited use of useful in preventing damage to normal tissues after the acti- peptides to a subset of HLA-restricted subjects (mainly + vation of CTLs by active immunotherapy. The agonist pep- HLA-A2 ) [15, 17]. tide can also have amino acid substitutions that enhance its Interesting results proposed by Huarte et al. showed that immunogenicity compared to the native sequence. Moreover, selective amino acid replacements could enhance the immu- the immunogenicity of the agonist peptide can be further nogenicity of a CTL epitope from CEA when it was used as enhanced by conjugating it to a helper peptide, to large car- a peptide-based vaccine in HLA-A2Kb-transgenic mice. The rier molecules, such as tetanus toxoid, poly-L-lysine, Pseu- authors observed that the replacement of the NH2–terminal domonas exotoxin A, or to an immunoglobulin molecule amino acid of the HLA-A2-restricted CTL epitope CEA691 specific for a surface receptor present on tumor cells and with tyrosine (Y) and the concomitant replacement of the absent on normal cells, thus allowing for selective targeting. valine (V) in position 7 with a leucine (L) (IMIGVLVGV The agonist peptide may be formulated into a pharmaceutical YMIGVLLGV) resulted in an improvement of CEA peptide composition in combination with a pharmaceutically accept- immunogenicity both in vitro and in vivo. These data open able carrier for use as an immunogen in a mammal [129, the possibility of using novel modified peptides derived from 130]. CEA to treat patients with CEA-expressing tumors [121]. Similarly, Kobayashi et al. generated a CEA-peptide Combined Delivery-based Vaccines from residues 653 to 667 (CEA653-667) that was able to induce Combined immunization is a good approach to enhance in vitro T-helper responses. In addition, the authors demon- the efficiency of vaccination with DNA, protein or virus strated that some of the peptide-reactive T-helper lympho- alone. cytes were able to recognize the CEA protein in the form of the recombinant protein or from tumor cells that express a) Prime DNA-Protein Boost Vaccination CEA [122]. Several strategies have been employed to enhance the Moreover, it has been demonstrated in vitro that the CEA potency of DNA vaccination. In this regard, immunization peptide CAP-1 and its agonist form induce the up-regulation with pKCEA66 in combination with the recombinant mGM- of different sets of pro-inflammatory genes in target T-cells. CSF (rmGM-CSF) adjuvant induced strong cellular re- + + In addition, the native peptide was also able to induce a sig- sponses mediated by CD8 and CD4 T cells. Moreover, the nificant up-regulation of the lymphotactin gene, which may heterologous pKCEA66 DNA prime/rCEA protein boost then be responsible for T-cell activation processes and sub- vaccination strategy resulted in increased humoral and cell- sequent anti-tumor effects [123-126]. mediated responses against hCEA in BALB/c mice [131]. A different strategy to deliver CEA antigen to the im- The efficiency of DNA vaccination could also be en- mune system has been recently proposed by Dai et al., who hanced using a late DNA boost instead of a heterologous collected exosomes from heat-stressed CEA0+-tumor cells protein boost. A vaccination protocol consisting of a prime (CEA+/HS-Exo) to provide both the antigen and the adju- vaccination with the pKCEA66 followed by a boost with vants (represented by exosomal heat shock protein-70 and wild type (wt) or recombinant CEA DNA fused with a teta- MHC-I) to the immune systems of recipient mice. The nus T helper epitope and delivered by electroporation has authors demonstrated that the intra-exosomal presence of been attempted. A total of 50% of the immunized mice both CEA and adjuvant proteins resulted in functional DC boosted with rCEA showed only an anti-CEA IgG response. maturation, which was likely responsible for the increased In contrast, a boost with wt CEA DNA led to an increase in + transferrable CEA-specific CTL response [127]. CD8 T cells, and it also induced high CEA-specific IgG titers in all immunized mice. Accordingly, it was suggested A vaccine comprising one peptide with less than 600 that the use of DNA for priming and boosting vaccination contiguous amino acids and 100% identity to a native se- could ameliorate the efficiency of anti-tumor vaccination quence of CEA is described in the US20030224036 patent CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 279 because it is a safer and stronger boost than rCEA, which Similarly, Peruzzi et al. immunized CEA.Tg mice with should reduce the time and costs of vaccination [132]. pV1J-CEA-LTB encoding hCEA and then boosted them with a recombinant Ad vector based on the chimpanzee sero- b) Prime DNA-virus Boost Vaccination type 3 (ChAd3) and engineered to express human CEA pro- rAd-expressing immunomodulatory molecules have also tein. They demonstrated that the ChAd3 vector was able to been used to improve the efficacy of DNA vaccination. A induce immune responses comparable to that of human Ad5- protocol of immunization using the combination between a based vectors without being hindered by a pre-existing im- recombinant adenovirus expressing HSP72 (ADHSP72) pro- munity to human Ads [138]. tein and a CEA-based DNA vaccine was used in C57BL/6 The promising results obtained from mouse models and mice. After immunization, these mice were inoculated with nonhuman primates suggested that the combined vaccination TrampC2-CEA cells, and 80% of the immunized mice strategy using DNA and Ad vectors expressing TAAs could showed a resistance to tumor formation and a long-term per- be a valid candidate for the development of new vaccines for sistent anti-tumor immune response mediated by anti-CEA the treatment of human cancers [136-138]. humoral (IgG production) and cellular responses (CTL activation). Moreover, all mice treated with the combined vacci- Indeed, the same authors patented polynucleotides encod- nation method showed a better reduction of tumor growth ing rhCEA and codon-optimized polynucleotides encoding and a more prolonged survival than mice immunized with rhCEA protein for high-level expression in human cells. the CEA-plasmid or with ADHSP72 alone [133]. Moreover, adenoviral and plasmid-based vectors comprising these synthetic polynucleotides have been provided for the Adenoviral vectors engineered to express the CEA anti- generation of immunogenic compositions and vaccines for gen as a fusion protein together with major immunostimulant treating CEA-associated cancers [139, 140]. molecules have also been employed. The results provided by Facciabene et al. showed that plasmid-carrying adenoviral Similarly, the use of adenoviral vectors and plasmid con- vectors encoding fusions of CEA and the minimized domain structs that carry codon-optimized human CEA in vaccines of tetanus toxin fragment C (Ad/CEA-DOM) or the B and pharmaceutical compositions for preventing and treating subunit of the heat-labile enterotoxin (Ad/CEA-LTB) were cancer has been described in another US patent application. useful when administered as a boost for the free plasmid The inventors provided synthetic DNA molecules encoding (called pV1J/CEA-DOM and pV1J/CEA-LTB, respectively). the human CEA protein, which were codon-optimized to This vaccination strategy induced significant CEA-specific allow for a high level of CEA expression in human host humoral and CTL responses, which protected C57BL/6 mice cells. In some embodiments of this invention, the recombi- from tumor growth after a challenge with MC38-CEA colon nant adenovirus vaccine was used in various prime/boost carcinoma cells [134, 135]. combinations with a plasmid-based polynucleotide vaccine to induce an enhanced immune response [141]. Aurisicchio et al. cloned the human CEA homolog (rhCEA) from rhesus monkey (Macaca mulatta) colon tissue In an European patent, the inventors describe polynucleo- samples and investigated whether plasmid DNA (pV1J- tides encoding a fusion protein comprising a portion of CEA rhCEA) and adenoviral vectors encoding rhCEA had immu- fused to a portion of an immunoenhancing element, such as a nogenic properties in mice and rhesus monkeys. These bacterial toxin. These polynucleotides were inserted into authors also created a codon-optimized rhCEA cDNA adenoviral vectors and into plasmid constructs. The recom- (rhCEAopt) to increase the level of expression of rhCEA and binant adenovirus vaccine was used in various prime/boost the immunogenic potency of the vectors expressing rhCEA. combinations with a plasmid-based polynucleotide vaccine The combined immunization strategy using DNA EP/Ad [142]. vectors expressing rhCEA or rhCEAopt showed immuno- c) Prime Virus-protein Boost Vaccination genic properties in C57BL/6 mice, eliciting humoral responses against rhCEA, which conferred protection against Bei and colleagues developed a strategy to potentiate the tumor growth. C57BL/6 mice immunized with rhCEAopt immunogenic activity of bV-CEA. The authors demonstrated vectors were also challenged subcutaneously with MC38- that the combined immunization based on priming with rV- CEA-2, and 57% of these animals remained tumor free. In CEA followed by a bV-CEA boost resulted in stronger im- addition, the authors demonstrated that vaccination with mune responses and anti-tumor activity than bV-CEA or rV- these vectors was able to break peripheral tolerance to CEA alone [143]. rhCEA in rhesus macaques, which developed both humoral and cell-mediated responses [136]. The authors investigated CEA-BASED ANTI-CANCER VACCINES: CLINICAL the immune response to human CEA after a combined vacci- TRIALS nation with DNA-EP/Ad vectors expressing human CEA Tsang et al. first demonstrated the possibility of evoking (hCEA) in murine models with intestinal tumors that over- CEA-specific cytotoxic T-cell responses to a specific CEA expressed CEA. Mice were immunized with the plasmid epitope mediated by a class I MHC-restricted mechanism in DNA (pV1J-CEA-LTBopt) encoding hCEA and then peripheral blood mononuclear cells (PBMCs) pulsed with boosted with an adenoviral vector expressing hCEA. After 50g/mL of CAP-1 peptide and 10U/mL of IL-2. Peripheral immunization, mice developed high titers of anti-CEA antibodies and a strong CD8+ T cell response, which conferred to blood lymphocytes (PBLs) were obtained from patients with metastatic carcinoma who were enrolled in a phase I trial mice the capacity to reduce tumor multiplicity and volumes based on vaccination with rV-CEA. PBLs were collected at different stages of tumor progression [137]. both before and after the administration of the vaccine [144]. 280 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al.

Preliminary results obtained in colon cancer patients im- tive responses to CEA were detected in 4 of 17 patients. No munized with recombinant CEA alone or with GM-CSF correlation between specific CEA-lymphoproliferative re- [145], anti-idiotypic antibodies mimicking CEA [146], or sponses and disease stabilization or marked declines in circu- recombinant vector-based vaccines such as ALVAC-CEA lating CEA could be demonstrated [155]. [147], rV-CEA [148], and ALVAC-CEA B7.1 [149], showed On the basis of previous results showing that vaccination the induction of cellular and humoral immune responses to with pKCEA66 (CEA 66 DNA) was able to elicit strong CEA, thus suggesting that CEA could be a suitable candidate humoral and cellular immune responses to CEA in mice to be used as a vaccine for colorectal cancer (CRC) patients. [21], a phase I open label controlled clinical trial in CRC CRC is the second most common type of malignancy in patients was performed by Staff et al. Ten patients with his- Western nations. More than 20% of patients with CRC pre- tologically confirmed stage II or III CRC entered into the sent with metastatic disease at the time of diagnosis [150]. study after resection of the primary tumor without evidence A total of 50% of CRC patients relapse, presumably be- of remaining macroscopic disease. Patients were divided into cause of the presence of minimal residual disease. Despite two cohorts. Cohort A received 10 intradermal injections of improvements in diagnostic and therapeutic methods, the 200g of CEA 66 DNA for a total dose of 2mg; cohort B global five-year survival in CRC has not substantially in- received four intramuscular injections of 2mg of vaccine for creased in recent years and remains approximately 50%. The a total dose of 8 mg. Patients received three cycles of vac- prognosis of patients with operable tumors depends on the cine at weeks 0, 2 and 6. Before the first vaccination, all pa- disease stage at the time of surgery. Five-year survival after tients received intravenous cyclophosphamide (300mg/m2) to surgery is 80-90% in Dukes A, 60-80% in Dukes B, 30-35% reduce the number of regulatory T-cells [156, 157]. All pa- in Dukes C, and 5-25% in Dukes D stage CRC [151]. Che- tients also received 150g of GM-CSF either intradermally motherapy based on combination treatment with 5-fluorou- or subcutaneously [145]. The treatment was well tolerated, racil (5-FU), CPT-11, and/or oxaliplatin represents the stan- and no grade 3 or 4 toxicity was observed. No clinical signs dard treatment for these patients. Currently, no other effec- of autoimmunity were reported. Seventy-two weeks after the tive treatment options are available after failure of these beginning of treatment, one patient in cohort A was deter- therapeutic regimens to improve the survival and quality of mined to have bladder cancer and died at week 99. One pa- life of these patients [152]. The use of a CEA-based vaccine tient in cohort B had disease recurrence at week 52. No evi- is considered a promising tool for the clinical treatment of dence of disease was observed in eight patients [158]. CRC patients because these patients show spontaneous hu- A phase I-II study based on CEA-mRNA and GM-CSF moral and cellular immune responses against TAAs includ- delivery was carried out by Rittig et al. on 30 stage IV renal ing CEA [153, 154], and a significantly better two-year sur- carcinoma patients. Patients received intradermal injections vival is linked with the presence of anti-CEA IgM antibodies of mRNA generated using plasmids coding for the tumor- [7]. associated antigens MUC1, CEA, Her-2/neu, telomerase, Accordingly, several different clinical trials in CRC pa- survivin, and MAGE-A1. Patients were divided into two tients, and also in patients with different neoplasms, have groups. The first group (n = 14) received 20g of vaccine on been performed that employ CEA as a vaccine using differ- days 0, 14, 28, and 42. The second group (n = 16) received ent modalities of CEA delivery. The outcomes of these clini- 50g of vaccine on days 0, 3, 7, 10, 28 and 42. Vaccinations cal trials are reported in Table 2. were repeated monthly in each group. The treatment was well tolerated in both groups. Six stable diseases (SD) and DNA-based Vaccines Alone or the Vaccine Plus Immu- one partial response were reported in the first group, and nostimulants nine SD in second group were observed. Median survival was 24 months in the first group and 29 months in second The safety and immunogenicity of a plasmid DNA vac- group. The induction of CD4+ and CD8+ T cell responses cine construct encoding CEA and hepatitis B surface antigen was also observed [159]. (HBsAg) in humans was evaluated by Conry et al. in a dose- escalation clinical trial on 17 patients affected by metastatic Anti-idiotype Antibody-Based Vaccines CRC expressing CEA and patients with an elevated serum CEA level. HBsAg was used to monitor the immune re- Foon et al. used MAb 3H1, which mimics a specific sponse to the vaccine. Escalating single and repetitive doses CEA-epitope, to treat 12 patients with advanced CRC refrac- of pCEA/HBsAg were administered to groups of three pa- tory to standard therapies. Patients received 1, 2, or 4mg of tients at total doses ranging from 0.1 to 6.0mg. The treatment aluminum hydroxide-precipitated anti-idiotype antibody in- was well tolerated, and only a few patients experienced tran- tracutaneously every week for a minimum of four intracuta- sient fatigue and creatine kinase elevation in a dose- neous injections. Idiotype-specific T cell proliferative re- independent manner. None of the patients produced anti- sponses were detected in 7 of 12 patients; four patients also double stranded DNA antibodies. However, no objective showed T cell proliferation in response to CEA. The treat- antitumor response was observed in 15 patients with measur- ment was well tolerated, with minimal toxicity limited to able disease. Five patients had stable disease at nine weeks local reaction, mild fever and chills. All patients experienced of follow-up, whereas 12 patients had disease progression. disease progression after 4-13 dosages of the vaccine [160]. HBsAg antibodies were induced in nine patients, whereas no The vaccination with an anti-idiotype antibody (CeaVac), detectable lymphoproliferative responses to HBsAg were which mimics CEA, was also employed by Foon et al. in a detected in any of the patients. None of the patients showed Phase lb clinical trial treating 24 patients affected by ad- CEA-specific antibody responses, whereas lymphoprolifera- vanced CEA-positive CRC. An anti-anti-idiotypic Ab3 CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 281

Table 2. CEA-based Cancer Vaccines: Clinical Trials.

Phase Number of Survival Type of Vaccine of the Evaluable Stage Anatomical Site Biological Effects CR PR NC PD Ref. (Months) Study Patients

DNA-BASED VAC- CINES ALONE OR VACCINES PLUS IMMUNOSTIMU- LANTS

Single/repetitive admini- CEA lymphoproliferative responses (4/17 stration of the CEA DNA I 17 IV Colorectal 0 0 5 12 NA [155] patients) vaccine

CEA66 DNA immuniza- I 10(a) II-III Colorectal No clinical signs of autoimmunity 0 0 8 2 NA [158] tion

mRNA encoding MUC1, 14(b) 0 1 6 3 24 CEA, Her-2/neu, telom- I-II IV Renal carcinoma CD4+ and CD8+ T cell responses [159] erase, survivin, MAGE- (c) A1 16 0 0 9 1 29

ANTI-IDIOTYPE ANTIBODIES-BASED VACCINES

Anti-CEA antibody (9 patients), idiotype IgG1 murine monoclonal specific T cell proliferative responses (7 [160] I 12 IV Colorectal 0 0 0 12 NA anti-idiotype antibody patients), in vitro CEA-specific T cell proliferation (4 patients)

Anti-idiotype Ab3 response (23 patients), [161] CeaVac I 24 IV Colorectal idiotypic T cell responses (10 patients), 0 0 0 24 11.3

CEA-T cell responses (5 patients)

Strong anti-CEA humoral and cellular im- CeaVac I 32 II-III-IV Colorectal 0 0 20 12 NA [146] mune response in all patients

Anti-CEA antibody responses (70%) and 5-FU/LV plus 3H1 III 412 14.7 3H1anti-Id response (Ab3) (63%)

IV Colorectal NA NA NA NA [162]

No anti-CEA antibodies, positive Ab3 5-FU/LV plus placebo 203 15.2 response (18%)

DC-BASED

VACCINES

Colorectal, breast, Pleomorphic infiltrates in DC injection sites CAP-1 pulsed PBL I 19 IV ovary, pancreas, 0 1(d) 1(e) 0 9 [163] (3 patients) ampulla of Vater

CAP1-6D IV or Colorectal, lung I 12 CD4+ and CD8+ T lymphocyte responses 1(f) 1 2(g) 7 NA [164] DCs recurrent

CEA652 pulsed DCs and I 10 IV Colon, lung, stomach DTH to peptide-pulsed DCs (2 patients) 0 0 2 8 NA [166] IFN- and TNF-

Lung, breast, colon Small increase of CEA-specific T cell activ- I 24 1 2 3 18 15.9 mRNA encoding CEA ity IV [167] transfected DCs Breast, colon, un- II 13 0 0 3 9 NA known Immunological responses 282 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al.

(Table 2) Contd….

Phase Number of Survival Type of Vaccine of the Evaluable Stage Anatomical Site Biological Effects CR PR NC PD Ref. (Months) Study Patients

HLAA* 0201- or HLA- A*2402-restricted CEA I 10 IV Colorectal CEA-specific T cell response 0 0 2 8 NA [152] peptides pulsed DCs

Colorectal, gallblad- CEA652 pulsed DCs I 8 IV Decrease of serum CEA levels (1 patient) 0 0 3(h) 8 NA [168] der

(i) Positive skin response and positive in vitro 12.9 CEA 652 pulsed DCs I 18 IV Gastrointestinal, lung 0 0 4 14 [169] CTL response

Colorectal, lung CEAalt peptide-specific IFN-gamma produc- CEAalt pulsed DCs I-II 9 IV 0 0 0 9 NA [170] cancer ing CD8+ T cells number increase

NK cell frequency and cytolytic activity CEA 652 pulsed DCs I 8 IV Colorectal 0 0 1 7 NA [171] increase (SD patient)

DCs pulsed with WT1, CEA, MAGE-1 and I 5 III -IV NSCLC Specific immune response improvement 0 2(l) 3(l) 5 2.7-9.2 [172] HER-2 peptides

DCs transfection with NA NA NA NA NA I 5 IV Colorectal No CEA-specific T cell CEA mRNA [174] vs 11 IV Colorectal CEA peptide-specific T cell (8/11 patients) peptide pulsed DCs NA NA NA NA NA

CD14+ precursors, pulsed IA, IB, Tumor-antigen independent response (5 with apoptotic bodies of IIB, I 16 NSCLC patients), NA NA NA NA NA [175] an allogeneic NSCLC IIIA, antigen specific response (6 patients ) cell line IIIB

7 I-II NSCLC Immunologic responses (6/7 patients) 6(m) 0 1(n) 0 NA

1650 cell line pulsed DCs I [177]

7 III NSCLC Immunologic responses (4/7 patients) 3(m) 0 1(o) 3 NA

Some patients showed specific immune Tumor Lysates pulsed responses. Partial or complete stabilization I 6 IV Colorectal 0 0 0 6 NA [178] DCs of serum CEA levels (4 patients)

Denileukin diftitox plus Decreased percentage of Treg cells at the DC-rF-CEA(6D)- I 15 IV Colorectal, breast 0 1 3 11 NA [183] end of the immunizations TRICOM

RECOMBINANT VECTOR-BASED VACCINES

Gastrointestinal, No primary T cell lymphoproliferative rV-CEA I 26 IV NA NA NA NA NA [184] lung, breast response

T cell lymphoproliferative response against rV-CEA I 5 III Colorectal NA NA NA NA NA [185] CEA (2 patients)

Colorectal, cholangiocarcinoma, gas- No CEA-specific lymphoproliferative re- rV-CEA I 20 IV 0 0 4(p) 16 NA [186] troesophageal, breast, sponses thyroid,

ALVAC-CEA I 8 IV Colon carcinoma CTL responses to CEA NA NA NA NA NA [188] CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 283

(Table 2) Contd….

Phase Number of Survival Type of Vaccine of the Evaluable Stage Anatomical Site Biological Effects CR PR NC PD Ref. (Months) Study Patients

Colorectal, breast, pancreas, appendix, ALVAC-CEA B7.1 Increases CEA-specific T cell response I 30 IV esophagus, gallblad- 0 0 8 22 NA [189] alone or with GM-CSF increase in vaccine alone group der, lung, thyroid

CEA-specific precursor T cells increase (3 CEA-expressing ALVAC-CEA-B7.1 I 18 IV SD patients) 0 0 3 15 NA [190] adenocarcinomas

Colorectal, lung, rV-CEA(6D)-TRICOM breast, thyroid, Enhanced CEA-specific T cell responses in I 58 IV unknown primary, 1(q) 0 23 34 NA(r) [195] most of the patients rF-CEA(6D)-TRICOM ovary, gastrointestinal

Colorectal, gastric, Prime (PANVAC-V)- pancreas, appendix, CEA-specific CD4+ responses (8 /16 pa- I 25 IV 0 2 3 NA NA(s) [200] Boost (PANVAC-V) esophagus, ovarian, tients) breast, lung

ALVAC followed by 34 chemotherapy and 50% Increases in CEA-specific T cells 0 15 14 5 NA booster vaccination

ALVAC and tetanus 32 II IV Colorectal 37% Increases in CEA-specificT cells 0 10 15 5 NA [201] toxoid followed by chemotherapy

38 Chemotherapy alone 30% Increases in CEA-specificT cells 2 15 13 5 NA

followed by ALVAC

Dose-dependent CEA elevation in peritoneal MV-CEA I 21 IV Ovarian 0 0 14 NA 12.15 [202] fluid and serum

No post-theatment increase in CEA-specific CEA/TRICOM I 12 I Gastrointestinal NA NA NA NA NA [204] T cells (t)

Breast 1 0 4 7 12.7(u) PANVAC I 26 IV Not evaluated [205] Ovarian 0 0 3 11 15 (v)

PROTEIN- AND PEP- TIDES- BASED VAC- CINES

rhCEA alone or in com- Strong rhCEA-specific proliferative T cell I 18 I-II-III Colorectal NA NA NA NA NA [145] bination with GM-CSF response in the GM-CSF group

Multi-antigen mixed Significant increase of lymphocyte prolifera- vaccine plus GM-CSF II 42 IV Breast NA 2 7 7 NA [206] tive responses and IL-2

rCEA alone or associated Colorectal Long-lasting anti-CEA-specific T cell and I 24 I-II-III NA NA NA NA NA [207] with GM-CSF IgG antibody response in GM-CSF group 284 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al.

(Table 2) Contd….

Phase Number of Survival Type of Vaccine of the Evaluable Stage Anatomical Site Biological Effects CR PR NC PD Ref. (Months) Study Patients

COMBINED VACCI-

NATION

Colon, esophagus, VAAA breast, cervix, un- VAAA induced a superior generation of I 18 IV known primary, 0 0 (z) (z) NA [147] CEA-specific T cell response AAAV stomach, rectum, pancreas

Patients receiving vaccine plus GM-CSF VAAA plus GM-CSF I 9 NA NA showed T cell counts significantly higher NA NA NA NA NA [208] and IL-2 than those treated with vaccine alone

PANVAC-V followed by Antibody responses against vaccinia virus in 3 administrations with I 10 IV Pancreas NA NA NA NA 6.3 [209] all patients PANVAC-F

CR= Complete Response; PR=Partial Response; NC= No Change; PD= Progression Disease; NA= Not Available. NSCLC= Non Small Cell Lung Cancer; (a) Cohort A received 10 intradermal injections of 200g of CEA 66 DNA for a total dose of 2mg; cohort B received four intramuscular injections of 2 mg of vaccine for a total dose of 8 mg; (b) 20g of vaccine on days 0, 14, 28, 42; (c) 50g of vaccine on days 0, 3, 7,10, 28, 42; (d) one patient with ovarian cancer had a minor response; (e) one patient with bone metastasis from breast cancer had stable disease; (f) this patient affected by metastatic CR had a complete resolution of pulmonary metastasis and malignant pleural effusion 4 months after vaccination. 10 months later she developed intraabdominal metastasis; (g) this clinical status lasted 3 months for one patient and 6 months for the other; (h) lasting for 3, 4, and 5 months, respectively; (i) value was calculated in 17 patients; one patient with CR and one with lung cancer were still alive after 31 and 46 months respectively; (l) response to conventional treatment; (m) no evidence of disease after surgical resection at the time of the report; (n) skeletal metastasis remained stable; (o) brain metastasis remained stable; (p) disease stabilization was observed in 4 patients (1colon, 1 thyroid, 1 rectal and 1 gastroesophageal); (q) patient with limited-stage small-cell lung cancer; (r) cohorts 7 and 8 that received the highest dose of vaccine with GM-CSF showed a survival advantage respect to cohorts 3 and 6 treated with the highest dose of vaccine without GM-CSF; (s) progression-free survival 2-30+ months, overall survival 6-30+ months; (t) immunological analysis was performed only in two patients; (u) median time to progression 2.5 months; (v) median time to progression 2 months; (z) see text.

response was detected in 23 patients. Ten patients had idio- incompletely resected Dukes’ D disease did not show evi- typic T-cell responses, and five had specific T-cell responses dence of progression after 14 months [146]. to CEA. The authors reported an overall survival of 11.3 Chong et al. conducted a phase III double-blind random- months. It is noteworthy that these results were comparable ized study with 630 patients with metastatic CRC to compare to those reported in other phase II trials with patients af- the clinical efficacies of the treatment with 5-FU/leucovorin fected by advanced CRC treated with various anticancer (LV) plus MAb 3H1 to treatment with 5-FU/LV plus pla- agents, including irinotecan, but with considerably less toxic- cebo. Fifteen patients did not receive treatment. Among the ity [161]. 615 treated patients, 412 received 5-FU and LV plus MAb Immune responses and clinical outcomes in response to 3H1 and 203 received 5-FU and LV plus placebo. A total of treatment with the CeaVac were also evaluated by Foon 70% of the patients treated with MAb 3H1 showed anti-CEA et al. in 32 patients with surgically resected Dukes' B (n = 4), antibody responses. The strength of the humoral response C (n = 11), and D (n = 8) stage CRC, and incompletely re- appeared to be associated with survival in the MAb 3H1- sected Dukes' D (n = 9) CEA-positive CRC patients without treated patients. No significant difference in tumor response measurable disease by CT scan. All patients were vaccinated was observed between the treatment arms. Survival was 14.7 with CeaVac with Alum or QS21 adjuvant. In addition, 14 of months for the Mab 3H1 arm and 15.2 months for the pla- these patients also received concomitant chemotherapy cebo arm (p = 0.80). A median survival of 8.3 months was treatment with 5-FU. High-titer immunoglobulin G and T- reported in the 102 patients with a negative CEA response (p cell proliferative immune responses to CEA were observed < 0.001). The treatment arms did not show significant differ- in all patients. No qualitative or quantitative differences in ences with one another with regard to tumor response [162]. the immune response were observed in patients receiving concomitant 5-FU. The authors demonstrated the ability to Dendritic Cells-based Vaccines break immune tolerance to CEA in 100% of the 32 treated patients. Disease progression was observed in 3 of 15 pa- Several clinical trials have been performed that employ tients with Dukes' B and C stage cancers after 19, 24, and 35 DCs pulsed with CEA peptides, CEA-mRNA, or tumor cells months, respectively. Among the patients with resected expressing CEA. Dukes' D disease, one relapsed after nine months and seven Morse et al. investigated the effectiveness of a vaccine remained free of evidence of disease at 12 and 33 months. based on peripheral blood DCs pulsed with CAP-1, which Eight of the nine patients with incompletely resected Dukes’ was administered intravenously every one or two weeks in a D disease progressed from 6 to 31 months. One patient with group of 21 patients affected by CEA-expressing metastatic CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 285 malignancies of different origins. A total of 19 patients were On the basis of the result obtained after the phase I study, evaluable for their clinical response (9 men and 12 women). Morse et al. conducted a phase II study of 13 patients with No toxicities directly related to the treatment were observed, evidence of disease after potentially curative surgery. and the treatment was well tolerated. One patient with ovar- Twelve patients (1 breast, 11 colon) were treated intrave- ian cancer had a minor response, and one patient with bone nously with 30x106 cells and 1x106 cells intradermally every metastasis from breast cancer had stable disease [163]. two weeks for four doses. One patient received only 1x107 cells intradermally. The authors observed immunological In a phase I study, Fong et al. demonstrated that toler- responses in the sites of injection of the DCs and in the pe- ance to CEA could be reversed by immunization with DCs ripheral blood of selected patients [167]. pulsed with the CAP1-6D peptide obtained by modifying the HLA-A2 CEA CAP-1 peptide [124, 125, 164]. They ob- To evaluate the safety of the treatment and the immune served CD4+ and CD8+ T lymphocyte responses to CEA in a response against CEA before and after DCs vaccination, Liu group of 12 patients expressing the HLA A*0201 allele who et al. enrolled 10 patients (6 with the HLA-A*2402 genotype were affected by metastatic or recurrent cancer (9 colon, 1 and 4 patients with the HLA-A*0201 genotype) affected by rectal, 1 lung). Four months after vaccination, a complete metastatic CRC who had failed a second-line chemotherapy response was observed in one patient affected by CRC with regimen based on CPT-11 or oxaliplatin. All patients were pulmonary metastasis and malignant pleural effusion. This vaccinated intranodally once a week for three weeks, fol- patient developed an intraabdominal metastasis 10 months lowed by a single booster two weeks later with DCs pulsed later. Another patient with colon cancer had a regression of with HLAA* 0201- or HLA-A*2402-restricted CEA pep- some, but not all, liver metastases. The other two patients tides. No grade II-IV toxicities or evidence of treatment- showed stable disease lasting three months and six months, related autoimmune response were detected. Only one pa- respectively [164]. tient developed a mild fever lasting 24 hours after the second On the basis of the previous observations reported by and third injections. Most patients (70%) had a substantial increase in the number of CEA-specific T cells, indicating a Nukaya et al., who identified a nonamer peptide (CEA652, boost in the CEA-specific T-cell response. Stable disease for TYACFVSNL) with the ability to induce in vitro restricted at least 12 weeks was reported in two patients, but most of HLA-A24 cytotoxic T lymphocytes [165], Itoh et al. con- them had progressive disease [152]. ducted a clinical study on ten patients affected by metastatic gastrointestinal or lung cancer with serum CEA levels rang- Matsuda et al. performed a pilot study on eight patients ing from 4 to 394 ng/ml to evaluate the safety and feasibility affected by advanced gastrointestinal malignancies to evalu- of vaccination based on CEA652 peptide-pulsed DCs and ate whether vaccination with CEA peptide-pulsed DCs could adjuvant cytokines (IFN- and TNF-). The toxicity of the enhance cytotoxic T-lymphocyte responses. Patients were vaccine was mild and not dose-related. No grade III or IV HLAA24-positive and had positive serum CEA levels (>5 toxicities were reported. After vaccination, two patients in ng/ml). All patients received CEA652 peptide-pulsed DCs the adjuvant arm showed immunological delayed-type hy- subcutaneously every two or three weeks. One patient had a persensitivity (DTH) to peptide-pulsed DCs based on a skin dramatic decrease of serum CEA levels. The other patients test, whereas no cases of DTH were observed in three pa- experienced disease progression, except three who had dis- tients who received the DC vaccine alone. The two patients ease stabilization lasting for 3, 4, and 5 months, respectively. with DTH had disease stabilization lasting for six and nine No adverse effects were observed except mild watery diar- months, respectively [166]. rhea in one patient on the day after the third injection [168]. In a phase I-II study, Morse et al. evaluated the effects of The feasibility and efficacy of vaccination with DCs immunization with DCs transfected with RNA encoding pulsed with the CEA652 peptide were also evaluated by CEA. The phase I dose-escalating study was carried out on Ueda et al. Eighteen patients with CEA-expressing metas- 29 patients who were affected by advanced cancers express- tatic gastrointestinal or lung cancer received intradermal and ing CEA (9 lung, 9 breast, 11 colon). Patients were divided subcutaneous treatments every two weeks. The treatment into three groups. The first group received 1x107 CEA was well tolerated, and most of the patients in whom treat- mRNA-transfected DCs (DC-CEA) intravenously weekly for ment was clinically effective showed a positive skin re- four weeks; the second group received 3x107 CEA mRNA- sponse to CEA652-pulsed DCs and a positive in vitro CTL transfected DCs intravenously along with an intradermal response to the CEA652 peptide [169]. dose of 1x107 DC-CEA every two weeks for four weeks; and Babatz et al. enrolled nine HLA-A*0201 patients with the third group received 1x108 DC-CEA and an intradermal 6 CEA-positive metastatic colorectal (n = 7) or lung cancer dose of 1x10 DC-CEA every two weeks for four weeks. The (n=2) in a phase I/II clinical trial to determine the capability last group also received a subcutaneous daily dose of 1.2x106 of DCs pulsed with a CEA-derived altered peptide (CEAalt) units of IL-2 for four days after DC-CEA administration. + to induce specific CD8 T cells in cancer patients. Patients Twenty-four patients were evaluable for response. One pa- received four doses of vaccine every two weeks (days 8, 22, tient with lung cancer not measurable with imaging studies 36, 50). The authors showed that vaccination induced an showed a complete response as assessed by a dramatic de- increase in the number of CEAalt peptide-specific IFN- cline in serum CEA levels to normal values. Two patients gamma producing CD8+ T cells in five of nine patients. had minor response, 3 patients showed stable disease lasting + CD8 T lymphocytes capable recognizing the native CEA more than 2 years and 18 patients underwent progression. peptide were detected in three of nine patients. However, The treatment protocol was well tolerated, and no acute tox- none of the patients had objective tumor regressions, and all icities related to DCs administration were observed. patients experienced disease progression. Seven patients 286 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al. showed progressive disease after the completion of four vac- broader immune response compared to CEA peptide-pulsed cinations [170]. DCs. Prior to surgical resection of metastases, patients received CEA-derived HLA-A2 binding peptide-loaded or A clinical trial with CEA peptide-loaded DCs generated CEA mRNA-electroporated DCs intravenously and in- with a combination of OK432 (Streptococcus pyogenes preparation), prostanoid, and interferon- (OPA-DC) was tradermally three times weekly. CEA peptide-specific T-cells were detected in 8 out of 11 patients in the peptide group. also conducted by Sakakibara et al. in ten patients affected None of the five patients in the RNA group showed CEA- by advanced CRC. One patient with stable disease (SD) and specific T-cells. The median progression-free survival was seven patients with progressive disease (PD) were reported 18 months (range 1-77 months) in the peptide-pulsed DC among the eight evaluable patients. An increase in natural group and 26 months (range 13-41 months) in the mRNA- killer (NK) cell counts and cytolytic activity was detected in the SD patient [171]. loaded DC group [174]. The tolerability and measurement of immunologic re- The effectiveness of peptide pulsed-DCs was also evalu- sponses to the allogenic pulsed-DCs were assessed in a non- ated by Perroud et al. in a non-randomized Phase I trial on randomized trial by Hirschowitz et al., who evaluated im- five HLA-A2 patients with inoperable, histopathologically munologic responses in a group of NSCLC patients treated confirmed non-small cell lung cancer (NSCLC) to verify whether the vaccination could consolidate the responses to with surgery, chemoradiation, or multimodality therapy. Six- teen patients with histologically confirmed stage IA (one conventional therapy. Four patients were stage IIIB and one patient), IB (4 patients), IIB (one patient), IIIA (7 patients), was stage IV at the time of the diagnosis. DCs were pulsed IIIB (3 patients) NSCLC received a prime vaccine and a sin- with WT1, CEA, MAGE-1, and HER-2 peptides. Patients gle boost one month apart intradermally. The DCs vaccine were submitted to computed tomography (CT) scans of the was generated using autologous CD14+ precursors pulsed thorax, abdomen and brain to evaluate the anti-tumor response after four cycles of chemotherapy with or without with apoptotic bodies of an allogeneic NSCLC cell line overexpressing Her2/neu, CEA, WT1, Mage2, and survivin. radiotherapy. Patients with PD were excluded from the 7 No clear immunologic response was detected in five patients. study. Patients received two doses of 510 DCs adminis- Five patients showed a tumor antigen-independent response, tered subcutaneously and intravenously two times at two and six developed an antigen-specific response. Immu- week intervals. An improvement in the specific immune re- nologic responses were not influenced by stage and prior sponse was detected after the immunization. One patient had a systemic reaction due to the treatment (grade 2 fatigue, therapy. Five patients experienced disease recurrence. One patient with stage IB NSCLC developed a solitary brain me- grade 3 leukopenia, grade 3 granulocytopenia, grade 3 lym- tastasis two months after the first vaccination. No evidence phopenia, grade 1 thrombocytopenia, grade 2 anemia and of disease was observed 15 months following resection of grade 3 hyponatremia). Two patients showed a partial re- the metastatic focus [175]. sponse to the conventional therapy, while three had SD. All patients had a clinical follow-up lasting a minimum of 8.5 In a nonrandomized trial, Hirschowitz et al. evaluated the months from the diagnosis and almost 3 months from the immunologic responses to immature, adenocarcinoma cell second dose of the vaccine. The time to the disease progres- line-pulsed autologous DCs in 14 patients with NSCLC. Pa- sion and survival from the last dose of the vaccine ranged tients were stratified by stage and prior therapy: surgically between 1 to 82 and 82 to 277 days, respectively. A longer resected I-II stage patients (n = 7) receiving or not receiving survival than expected for their TNM stage was observed in adjuvant therapy vs. unresectable stage III patients (n = 7) three patients. Two of these patients were the only ones with treated with chemo-radiation alone. DCs were pulsed with HER-2- and CEA-positive tumors [172]. the adenocarcinoma cell line 1650 overexpressing Her-2/neu, CEA, Mage 2, WT-1 and survivin [175-177]. Patients re- Conventional chemotherapy followed by immunotherapy ceived DCs intradermally as the prime vaccine and a single using DCs and cytokine-induced killer (CIK) cells for the boost one month later. No adverse events related to treatment treatment of stage IIIB to stage IV NSCLC was evaluated by were reported. Immunologic responses were seen in 4/7 Zhong et al. Twenty-eight untreated patients entered into the study. Fourteen patients received conventional vinorelbine- stage III unresectable and 6/7 stage I/II surgically resected patients. Recurrence was reported in one of seven surgically platinum (NP) chemotherapy alone, and 14 patients received resected patients. Four of seven stage III patients experi- CEA (605-613) peptide-pulsed autologous DCs and CIK enced disease progression. No immunologic response was cells after NP chemotherapy at 30-day intervals for four cy- observed in three of five patients with progressive disease. cles. The adverse effects resulting from the chemoimmuno- Radiographically confirmed SD at 23 and 51 months follow- therapy were mild and tolerable. The chemoimmunotherapy group showed a significantly prolonged time to progression ing initial chemo/radiation was reported in 2 patients with unresectable stage III disease who had an immunologic re- with respect to patients in the chemotherapy group (median sponse to immunization. In the unresectable stage III group, improvement from 5.2 months to 6.9 months (95% CI: 5.0- a case of stable disease was also reported in one patient 8.8). The 1-, 2-, and 5-year survival rates were 64.3, 49, and without an immunologic response [177]. 21.0%, respectively in the chemoimmunotherapy group. However, no significant difference in the overall survival The safety and effectiveness of tumor lysate (TL)-pulsed rate was observed [173]. DCs have been evaluated by Tamir et al. in six HLA- A*0201-positive patients affected by CEA-positive CRC. Sixteen patients with resectable liver metastases of CRC The treatment was well tolerated. Some patients showed were enrolled by Lesterhuis et al. to evaluate whether the specific immune responses. Partial or complete stabilization transfection of DCs with CEA mRNA could induce a CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 287 of serum CEA levels was observed in four patients, but no sophageal cancer). Most of the patients (17 of 20) had ele- clinically objective responses were reported [178]. vated serum CEA levels at the time of their entry in the study. None of the patients showed CEA-specific lymphop- Based on previous studies that demonstrated that CD4- roliferative responses, although disease stabilization was CD25 highFoxP3-regulatory T (Treg) cells were able to limit antigen-specific immune responses and suppress antitumor observed in four patients (1 colon, 1 thyroid, 1 rectal and 1 gastroesophageal). A total of 16 patients experienced disease immunity [179-182], Morse et al. conducted a pilot study on progression [186]. 15 patients with advanced CEA-expressing malignancies (14 colorectal cancer and 1 breast cancer). The aim of this trial The safety of the ALVAC-CEA vaccine was first demon- was to evaluate if Tregs depletion could enhance immune strated by Marshall et al. in a phase I study of 20 patients responses against a cancer vaccine consisting of DCs modi- affected by advanced CEA-expressing tumors. Seven pa- fied with the viral vector rF-CEA(6D)-TRICOM vaccine. tients received ALVAC-CEA at doses of 2.5x105 pfu, seven Patients received denileukin diftitox before vaccination to at doses of 2.5x106 pfu, and six at doses of 2.5x107 pfu. The reduce the frequency of Tregs in the peripheral blood. vaccine was well tolerated at all dose levels without signifi- Denileukin diftitox is able to bind to cells expressing high cant toxicity. After vaccination, an increase in CEA-specific levels of CD25 and, after internalization, leads to their cell CTL precursors was observed in seven of nine HLA-A2– death. In cohort 1, nine patients received one dose of positive patients. No objective clinical antitumor responses denileukin diftitox (18g/kg) intravenously four days before were described. All patients experienced PD. The time to initiating DCs injections. DCs were administered every three progression was found to be 1-15 months [187]. weeks for four immunizations. To obtain a better Treg cells Using pre and postvaccination PBMCs obtained from depletion, patients in cohort 2 received denileukin diftitox heparinized blood of patients positive for HLA-class I A2 (9g/kg) intravenously four days before every DCs injection. alleles with metastatic colon carcinoma enrolled in the Phase In this clinical trial, the authors first demonstrated that mul- I trial performed by Marshall et al. [184], Zhu et al. demon- tiple administrations, but not a single administration, of strated the induction of CTL responses to CEA in seven of denileukin diftitox are able to deplete CD4+CD25highFoxP3+ eight patients after their vaccination with ALVAC-CEA. In Treg cells. An enhanced T-cell response to CEA was ob- addition, CTLs were able to lyse allogeneic and autologous served in the multiple-dose denileukin diftitox group, but not tumor cells in an MHC-restricted manner [188]. the single-dose denileukin diftitox group. These data indicated that the Tregs depletion strategy could enhance tumor A clinical trial employing the ALVAC-CEA-B7.1 vac- antigen-specific immune responses induced by vaccine cine was conducted by von Mehren et al. on 39 patients with treatment. Three patients had SD, and one had a minor re- advanced or metastatic CEA-positive tumors that were response to treatment. In cohort 1, one of the nine patients had fractory to standard therapy (22 colon, 6 rectal, 3 breast, 3 a minor response and one had SD; the remaining seven pa- pancreas, 1 appendix, 1 esophagus, 1 gallbladder, 1 lung, and tients showed PD. Another patient of this cohort who ini- 1 thyroid). Patients received either vaccine alone or the vac- tially had PD later showed disease stabilization. All six pa- cine with GM-CSF. In the initial dose escalation phase, the tients in cohort 2 experienced disease progression, although first group of three patients received 2.5x107pfu of ALVAC- one of these patients showed disease stabilization lasting for CEA-B7.1, whereas the second group received 1x108pfu. more than six months on subsequent CT scans [183]. The 30 remaining patients were treated with the working dose of 4.5x108 pfu. The vaccine was administered intrader- Recombinant Vector-based Vaccines mally every two weeks for a total of four injections. The treatment was well tolerated by most patients. Eight cases of Hamilton et al. utilized rV-CEA for the first time in a stable disease were observed among the 30 patients receiving phase I clinical trial approved by the National Cancer Insti- four cycles of vaccine and evaluable for their clinical re- tute (NCI) Institutional Review Board. This study was per- sponses. In six patients with SD, a decline in serum CEA formed on 26 patients with metastatic carcinoma (gastroin- levels lasting from 4 to 12 weeks was observed. Increases in testinal, lung, and breast). No primary T-cell lymphoprolif- the CEA-specific T-cell response were also reported after erative responses were detected in stimulated PBLs obtained four vaccinations in patients treated with the vaccine alone, prior to or after rV-CEA vaccination with soluble CEA pro- but not when combined with GM-CSF. Among the patients tein [184]. with SD, seven with CRC and one with pancreatic cancer A trial using rV-CEA was conducted by Conry et al. on received one to seven boost injections and continued to re- five patients with surgically resected Dukes’ C stage colorec- main stable. No partial or complete responses were observed tal carcinoma who had completed adjuvant chemotherapy. [189]. All patients received two doses of rV-CEA with minimal Another study in patients with advanced CEA-expressing toxicity at the injection site. After vaccination, two patients adenocarcinomas was conducted by Horig et al. to determine showed a T-cell lymphoproliferative response against CEA the effectiveness of the ALVAC-CEA-B7.1 vaccine. Patients [185]. were divided into three cohorts of six patients each. Each CEA-specific immune responses and antitumor effects cohort received increasing doses of the ALVAC-CEA-B7.1 6 7 8 induced by rV-CEA vaccination and the safety and relative vaccine (4.5x10 , 4.5x10 , and 4.5x10 pfu) by intramuscular efficacy of intradermal versus subcutaneous administration injection every four weeks for three months. SD correlated were also evaluated by Conry et al. in a randomized phase I with increasing CEA-specific precursor T cells was reported trial of 20 patients with metastatic disease (10 colon, 4 rectal, in three patients [190]. 2 cholangiocarcinoma, 1 thyroid, 1 breast and 1 gastroe- 288 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al.

On the basis of previous experimental studies in animal CEA-specific T-cell responses induced by the ALVAC- models showing that the addition of a triad of costimulatory CEA-B7.1 vaccine. Patients were randomized to receive 1) molecules (B7-1, ICAM-1, and LFA-3) to recombinant vac- the vaccine followed by chemotherapy and booster vaccina- cinia and avipox vectors expressing CEA enhances both tion (39 patients); 2) the vaccine and tetanus toxoid followed CEA-specific T-cell immunity and antitumor immunity [76- by chemotherapy (40 patients); or 3) chemotherapy alone 79, 89, 189, 191-194], Marshall et al. conducted the first trial followed by the vaccine in patients without disease progres- in humans using rV-CEA(6D)-TRICOM and rF-CEA(6D)- sion (39 patients). Toxicities were mainly related to the ex- TRICOM vaccines alone or in combination. The study was pected chemotherapy side effects. An objective clinical re- conducted with 58 patients with advanced CEA-expressing sponse was observed in 42 (40.4%) of the 104 evaluable tumors (35 colorectal, 9 lung, 3 breast, 1 thyroid, 2 unknown patients. Two complete responses were reported in group 3. primary tumors, 1 ovary, 7 other gastrointestinal), divided Forty objective partial responses were also reported (44.1% into eight cohorts. The study supported the previous observa- in group 1, 31.3% in group 2, and 39.5% in group 3). An tion that CEA-based vaccines can induce CEA-specific im- additional 42 patients (37.5%) equally distributed across all mune responses and antitumor activities in patients with ad- groups had SD. No significant differences in immune re- vanced carcinomas. Cohorts 1 to 3 were treated with a dose sponses between the treatment groups were observed. These escalation of rF-CEA(6D)-TRICOM. Cohorts 4 to 6 were findings suggested that chemotherapy did not affect the gen- treated with a dose escalation of rV-CEA(6D)-TRICOM and eration of CEA-specific T-cell responses following vaccina- the maximum tolerated dose of rF-CEA(6D)-TRICOM. Co- tion [201]. hort 7 received the same doses of vaccines administered to An Edmonston vaccine strain of MV engineered to ex- cohort 6, except that recombinant GM-CSF protein (100g) press CEA (MV-CEA) was used by Galanis et al. to treat 21 was administered subcutaneously into the vaccine injection patients with normal CEA levels affected by taxol and plati- sites on days 1 (day of first vaccination) through 4. The ma- num-refractory recurrent ovarian cancer in a phase I trial. jority of patients showed enhanced CEA-specific T-cell re- Patients received MV-CEA intraperitoneally every four sponses. Decreased or stable serum CEA levels were ob- weeks for up to six cycles at seven different dose levels. No served in 11 patients. One patient had a pathological re- dose-limiting toxicity was observed. A total of 14 of the 21 sponse. SD lasting at least four months was reported in 24 patients were observed to have dose-dependent disease stabi- patients who received at least four administrations of the lization with a median duration of 92.5 days. Patients also vaccine. One patient had SD lasting 4 months after two vac- showed an increase in median survival (12.15 months) with cinations. The authors concluded that TRICOM-CEA pox respect to the median survival of six months expected in this vaccines are safe when used alone or in combination in pa- patient population [202]. tients with advanced cancer [195]. On the basis of previous studies demonstrating that non- On the basis of previous clinical trials demonstrating that lethal doses of radiation were able to alter the phenotype of vaccines directed against MUC-1 or CEA are safe and able tumor cells and facilitate immune-mediated killing [203], to generate immune responses [144, 147, 196-199], Gulley Gulley et al. conducted a pilot study of 12 patients with et al. performed a pilot study based on the use of a poxviral CEA-positive gastrointestinal cancers metastatic to the liver vaccine expressing CEA and MUC-1, along with TRICOM to study the effects of a treatment based on CEA/TRICOM (designated PANVAC), engineered into vaccinia (PAN- vaccination in combination with low-dose radiation therapy. VAC-V) as a prime vaccination and into fowlpox (PAN- The vaccine was administered on day 1, followed by bi- VAC-F, generated using a replication-defective fowlpox weekly boosters and split-course radiation (32 Gy total) virus) as a booster vaccination. Twenty-five patients affected starting on day 21. The protocol was well tolerated, with by CEA- or MUC-1-expressing metastatic carcinomas en- limited evidence of clinical activity [204]. tered into the study (10 colorectal, 3 gastric, 2 pancreatic, 2 appendiceal, 1 esophageal, 3 ovarian, 2 breast, 2 lung). Nine The results of a study employing the PANVAC treatment of the 15 patients analyzed showed immune responses to regimen have also been reported by Mohebtash et al. MUC-1 and/or CEA after vaccination. CEA-specific CD4+ Twenty-six patients with progressive disease for metastatic responses were detected in 8 of 16 patients analyzed. One breast (n = 12) or ovarian cancer (n = 14) were enrolled to patient with stage IIIc clear cell ovarian cancer and sympto- evaluate the clinical outcomes of the vaccination. All pa- matic ascites had a durable (18-month) clinical response. The tients received 2x108pfu of PANVAC-V subcutaneously as a absence of ascites was confirmed at the first restaging. One priming vaccination, followed by PANVAC-F on days 15, patient with metastatic breast cancer and multiple bulky liver 29, and 43 and every 28 days thereafter. Patients also re- metastases had a 21% reduction of the lesions from baseline, ceived GM-CSF to increase the migration and maturation of and immune responses were seen to both CEA and MUC-1. APCs [90, 147, 205]. Three patients with ovarian cancer Lesions decreased by 24% from baseline at the second re- showed disease stabilization lasting from four to six months. staging and returned to baseline at the third restaging. No The median overall survival was 15.0 months. Among pa- evidence of disease for 18 months or more was reported in tients with breast cancer, one complete response was re- three patients without radiographic evidence of disease at ported; four patients had SD; and seven patients had PD. The beginning of the trial [200]. median overall survival was 13.7 months. The median time to progression was 2.5 months in breast cancer patients and A multi-institutional randomized clinical trial on 118 two months in ovarian cancer patients. A comparative analy- patients with metastatic CRC was conducted by Kaufman sis of CD8 immune response after in vitro stimulation could et al. to evaluate the effects of standard cytotoxic chemo- not be done due to the limited pre-vaccination sample [205]. therapy (5-FU, LV, and irinotecan) on the generation of CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 289

Protein- and Peptide-based Vaccines breast, 1 cervix, 1 unknown primary, 1 stomach, 1 rectum, 1 pancreas) and aimed to evaluate the safety of rV-CEA (V) Samanci et al. immunized a group of 18 patients affected and the avipox-CEA vaccine and the effects of the sequence by colorectal carcinoma who did not have evidence of mac- of administration on T-cell response. Patients were randomly roscopic disease after surgery with a recombinant source of assigned into two cohorts. The first cohort of nine patients human CEA (rhCEA) alone (n=9) or in combination with received one vaccination with rV-CEA followed by three GM-CSF (n = 9). All patients in the GM-CSF group showed boosts with avipox-CEA (VAAA). The second cohort of a strong rhCEA-dose-dependent IgG antibody response, nine patients received three vaccinations with avipox-CEA whereas only three of the patients in the rhCEA alone treat- followed by a vaccination with rV-CEA (AAAV). The vac- ment group showed a weak antibody response. A strong 7 cine was administered every 28 days. rV-CEA (1.0x10 pfu) rhCEA-specific proliferative T cell response was detected in was administered intradermally into the deltoids. Avipox- the GM-CSF group, whereas the rhCEA alone treatment CEA (2.5x107pfu) was administered subcutaneously. Pa- group had a weak rhCEA-specific T cell response [145]. tients also received GM-CSF (100g) as close to their most Jiang et al. performed a phase II study on 42 breast can- recent vaccination sites as possible, beginning on the day of cer patients using a multi-antigen vaccine that included the the avipox-CEA vaccination and for three consecutive days following antigens: autologous breast cancer cells (AUTOC), thereafter. The treatment with rV-CEA followed by avipox- allogeneic breast cancer MCF-7 cells (ALLOC), the TAAs, CEA induced a higher number of CEA-specific T-cell re- CA15-3, CEA and CA125, and low doses of GM-CSF and sponses in all six HLA-A2-positive patients. In contrast, only IL-2. Subcutaneous doses of the vaccine were administered two of six HLA-A2-positive patients of the second cohort at the 1st, 2nd, 3rd, 7th, 11th and 15th weeks. After vaccination, showed an increase in CEA-specific T-cell precursors. Some a significant increase in lymphocyte proliferative responses of the treated patients also demonstrated antibody production to AUTOC, CA15-3, CEA and CA125, with respect to pre- against CEA. No objective antitumor responses were ob- vaccination levels, was observed. One patient showed a re- served. One patient in the VAAA cohort showed a complete duction in the size and number of hepatic metastases; another radiographic response to fluorouracil for metastatic colon patient showed a decrease in the size of some osteolytic me- cancer to the liver and remained free of any evidence of dis- tastases. Clinical responses were evaluated in 16 patients ease for more than 21 months after the beginning of the vac- with stage IV breast cancer who had measurable disease. cination protocol. Another patient in the VAAA cohort who Seven patients (44%) had SD, and seven patients (44%) had had surgically resected liver metastases from colon cancer PD. No significant reduction of the serum tumor markers had a positive positron-emission tomography scan of ab- CA15-3, CEA and CA125 was observed [206]. dominal nodes more than 20 months after the beginning of vaccination. In the same cohort, one patient with surgically Ullenhag et al. performed an open-labeled, randomized, resected colon cancer without evidence of disease at the be- dose-ranging study on 24 patients (14 males and 10 females) ginning of the vaccination protocol underwent PD after 10 with CRC (17 with colon cancer, and 7 with rectal cancer) months. One patient with pseudomyxoma peritonei with me- who were previously treated with surgery and who had no evidence of macroscopic disease at the start of the immuni- tastasis in the lungs and abdomen remained stable for 6 months and experienced a minor reduction in the size of the zation treatment. Only one of the patients had received che- abdominal nodes. In addition, the CEA level decreased after motherapy after surgery. Groups of six consecutive patients IL-2 was added. In the VAAA cohort, two patients without were immunized seven times with rCEA at four different evidence of disease at the beginning of the treatment, one dose levels (100, 316, 1000, or 3000g). Patients in each with colon cancer and the other with rectal cancer, under- dose level group were randomized to receive seven immunizations with rCEA alone or rCEA with GM-CSF. Twenty went PD after 8 and 10 months, respectively [147]. patients completed the vaccination protocol. Four patients Marshall et al. continued the previous study in a phase I relapsed and were withdrawn from the protocol before the trial to evaluate the effects of adding GM-CSF and IL-2. entire vaccination schedule had been completed. Immu- Patients treated with the vaccine plus GM-CSF showed T- nological monitoring of patients lasting 36 months showed cell counts that were significantly higher than those treated that vaccination with rCEA plus GM-CSF induced a strong, with vaccine alone. Treatment with IL-2 did not produce T- long-lasting anti-CEA-specific T-cell response and an anti- cell counts higher than those observed with the vaccine alone CEA IgG antibody response. The study showed that patients [208]. who developed sustained high anti-CEA antibody titers had a Kaufman et al. conducted a phase I study based on re- significantly longer survival, thus providing evidence that combinant prime-boost poxviruses using the PANVAC. Ten CEA can be used successfully as a tumor antigen target. No patients affected by advanced pancreatic cancer received a statistical link between the proliferative T-cell response and treatment consisting of a priming vaccination with PAN- survival was observed in this study [207]. VAC-V followed by three administrations with PANVAC-F. Patients also received GM-CSF after each vaccine injection Combined Delivery-based Vaccines and for three consecutive days thereafter. Treatment was On the basis of preclinical evidence showing that the rV- repeated monthly up to 12 months. All 10 patients showed CEA and avipox-CEA vaccine combination, which utilizes a antibody responses against vaccinia virus. Antigen-specific T diversified prime-and-boost vaccination protocol, could gen- cell responses were observed in five patients. The median erate a more vigorous T-cell response than either vaccine overall survival was 6.3 months. Patients who generated anti alone [89], Marshall et al. performed a phase I study in 18 CEA- and/or anti-MUC-1-specific immune responses had a patients with advanced cancer (12 colon, 2 esophagus, 2 290 Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 Turriziani et al. significant increase in their overall survival with respect to body-based vaccines have the benefit of targeting a unique those who did not (15.1 vs. 3.9 months, respectively) [209]. tumor-specific antigen, although their production is very arduous. Peptide-based vaccines offer the advantage of being CURRENT & FUTURE DEVELOPMENTS immunogenic and safe, however they have potential drawbacks, such as the ability to target only one or few epitopes, Although in recent years, novel therapeutic approaches or the limited use of peptides to a subset of HLA-restricted have been developed to treat cancer, cancer is still one of the subjects [15, 17, 210]. most frequent causes of death in industrialized countries. In this regard, the treatment of most forms of cancer, especially Formulations and methods for enhancing vaccination in advanced stages, with conventional therapies such as sur- efficacy and potency in both animals and humans have been gery, chemotherapy and radiotherapy, is unsuccessful in patented, and several clinical trials have been performed. many cases. Immunotherapy represents an additional thera- The results from clinical trials have shown that these vacci- peutic tool for cancer treatment. The goal of cancer immuno- nation protocols are well tolerated by cancer patients and that therapy is to break the immune tolerance to TSAs and TAAs, they are safe. Despite the fact that vaccination was shown to thus inducing a strong immune response to these antigens. induce a strong immune response to CEA, resulting in a de- The ability of tumor cells to escape immunosurveillance, lay in tumor progression and prolonged survival in some particularly by inducing immune tolerance against tumor cancer patients, it failed to eradicate the tumor in most cases. antigens, prevents the host from efficiently counteracting Indeed, the efficacy of cancer vaccines is strongly re- tumor growth. In fact, despite the fact that TAAs such as duced by the negative effect exerted by the tumor microenvi- CEA are abundantly expressed on the surface of tumor cells, ronment on immune response. Immunosuppressive immune immune responses against these antigens are weak, and this cells, such as Tregs, suppressor CD8+ T cells, regulatory phenomenon represents one of the major obstacles to devel- natural killer cells and tumor associated macrophages, are oping an efficient cancer vaccine [15]. generated in the tumor microenvironment. Accordingly, an- Thus, the identification of an appropriate tumor antigen tigen-specific T cells are inactivated and their ability to kill as a target for immunotherapy represents one of the most tumor cells is lost [210]. On the other hand, one of the most important steps in the development of an effective cancer promising clinical approaches to overcome this drawback is vaccine. Several studies have shown that CEA can be envi- the use of a CEA-based vaccine in combination with immu- sioned as a target for cancer immunotherapy protocols. It nosuppressive cells inhibitors. For example, when the CEA- was previously demonstrated that tolerance to CEA could be TRICOM recombinant poxviruses were administered in interrupted by mouse immunization with recombinant human combination with Ipilimumab, a fully human monoclonal CEA [10] without leading to autoimmune disease [11, 12]. antibody against CTLA-4, an enhancement of antitumor ef- Animal models played a crucial role in understanding the fects of these vaccines has been observed [83, 211]. mechanisms of tumor immunology, and in the last decades, In addition, it should be noted that in many solid tumors, different modalities of immunization and experimental mod- the presence of a pronounced lymphoid infiltration is associ- els have been employed to determine whether CEA immuni- ated with a better prognosis. In this regard, the number of zation could induce an immune response that is able to ham- CD8+ T cells and a high ratio between CD8+ and CD4+ T per the growth of tumors expressing CEA [13-17]. The cells have been correlated with a better overall survival in promising results obtained from animal models provided the CRC patients [212]. In the light of these observations it ap- impetus for using the different strategies of immunization in pears that an ideal cancer vaccine, useful for the treatment of the treatment of tumors expressing CEA in humans. colorectal cancer, requires the ability to induce antigen- + Among the diverse CEA-based cancer vaccines, DCs- specific CD8 T cells which induce tumor cell lysis and anti- + and recombinant viruses-based vaccines seem the most valid. gen-specific CD4 T cells which release cytokines that en- DCs are the most effective APCs and play a crucial role in hance CTL activity [210]. inducing immune responses against tumors by priming T-cell Accordingly, further research is required to better eluci- responses to target TAAs. The use of DCs loaded with CEA date the effects of cancer vaccines on the innate and adaptive was shown to enhance CEA presentation to the host immune immunity to develop new vaccine scheduling and to improve system. Recombinant viruses-based vaccines present several the use of immunotherapy in clinical practice. Thus, in order advantages as compared to other delivery systems. For ex- to develop more efficient and potent cancer vaccines, it will ample, TAAs delivered by these vectors are more immuno- be necessary to design new clinical trials combining cancer genic than TAAs administered alone or in combination with vaccines with conventional therapies such as chemotherapy, adjuvants [70]. Moreover, certain recombinant viruses-based radiotherapy and drugs which target those factors responsible vaccines may be used for multiple immunizations without for immunosuppression of immune cells to better prevent risk of side effects. Besides, they have the ability to accept and treat CEA+ tumors. large fragments of foreign DNA, and thus may contain multiple genes such as TAAs, cytokines and co-stimulatory ACKNOWLEDGEMENTS molecules. In this regard, the use of recombinant viral CEA- TRICOM vectors provided the best antitumor effects [210]. The authors’ work was partly supported by Ministero Although DNA-based vaccines have been shown to induce dell’Istruzione, dell’Università e della Ricerca (MIUR, both humoral and cell-mediated immune responses against PRIN) (R.B). The authors wish to thank Jane Marie Gher- CEA in preclinical models, results from clinical trials have ghetta for manuscript editing. not been encouraging [155, 158, 159]. Anti-idiotype anti- CEA-Based Cancer Vaccines Recent Patents on Anti-Cancer Drug Discovery, 2012, Vol. 7, No. 3 291

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