Carcinoembryonic Antigen (CEA)

32 Pages·2012·0.46 MB·English

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 tu-
mor 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 im-
mune 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
CEA-BASED ANTI-CANCER VACCINES: MOUSE
immune responses in cancer patients. However, autoantibod-
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 pre-

viously 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: [phone]; Fax: [phone];
E-mail: [email protected] Since then, different modalities of immunization and
#These authors contributed equally to this work(cid:1)(cid:1) experimental models to determine whether CEA immuniza-
(cid:21)(cid:21)(cid:20)(cid:21)(cid:16)(cid:22)(cid:28)(cid:26)(cid:19)/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
mice immunized with pKCEA66. Moreover, the boost with
In addition, to enhance the potency of vaccination, com-
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 phe-
nomenon 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 (triCEA )was used to immunize BALB/c
625-667
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 engi-
neered 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 hu-
man 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-(cid:1), 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-(cid:1)
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-(cid:1) 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-(cid:1) 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-(cid:1) incidence (80%)
hCEA/DNA (VR-CEA) plus B7.1/IgG/DNA Suppression of tumor
CEA/LLC cells in C57BL/6 mice CTLs , IFN-(cid:1) [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-(cid:1) [36]
(VR-B7-1wa/Ig) incidence (60%)
hCEA/DNA (pCEA) plus mGM-CSF/DNA IgG1, IFN-(cid:1), 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%)
(cid:1)
(cid:1)
(cid:1)
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-(cid:1)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-(cid:1) 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-(cid:2) ,IL-5 growth (100%)
Regression of tumor
Ad5 [E1-, E2b-]-CEA MC38-CEA-2 cells in C57BL/6 mice IFN-(cid:2) 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-(cid:2) free survival time (80% of [98]
mice)
CTLs, antibodies, Regression of tumor
PV-CEA RMA-CEAFL cells in CEA tg mice [101]
IFN-(cid:2) 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]
(cid:1)
(cid:1)
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]
antibodies, IFN-(cid:1)
pKCEA66 plasmid in combination with
rCEA protein
Prime (hCEA /DNA)-boost (rCEA/protein)
Protective immunity
BALB/c mice IgG [132]
(50%)
pKCEA66 plasmid in combination with
rCEA protein
Prime (hCEA/DNA)-boost (hCEA/DNA)

Protective immunity
BALB/c mice IgG, CTLs, IFN-(cid:1) [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-(cid:1) 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-(cid:1)
of mice)
Prime (rhCEA/DNA)-boost (AdV-CEA)
Antibodies, CTLs, Suppression of tumor
MC38-CEA cells in C57BL/6 mice [136]
IFN-(cid:1) growth (57%)
pV1J-rhCEA in combination with Ad5-
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(cid:1)

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 CEA peptide and SM360320 led to a significant anti-tumor effect, which
691
(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 con-
The use of anti-idiotype (anti-Id) antibodies to induce
firming the potency of oral DNA vaccination as a candidate
TAA-specific humoral responses has been explored. Several
for human vaccine development [32].
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
QS-21 in the anti-idiotype (anti-Id) antibody vaccine was
ing tumor growth [33].
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
mice. These results suggested that CpG-ODN (1826) is an
Moreover, it has been demonstrated that co-delivery of a
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
In the US7090842 patent, the same inventors also dis-
by binding to CTLA-4 on T cells. When this plasmid is used
closed the design of diagnostic kits and other methods of
as a vaccine, the CTLA-4/B7-1 binding during T cell/T cell
using 3H1 polynucleotides, including methods of treating
interaction is blocked. This blockage leads to the inhibition
CEA-associated tumors with the 3H1 antibody. In this pat-
of negative regulatory signals, thus improving anti-tumor
ent, polynucleotides encoding polypeptides with the immu-
immunity. Mice vaccinated with the plasmid encoding hu-
nological activity of the monoclonal anti-Id antibody 3H1
man CEA and mutant B7-1/Ig showed a reduced tumor inci-
are described. These polynucleotides encode sequences of
dence of 60% [36]. Similarly, a fusion DNA plasmid encod-
both the variable light and heavy chain of the 3H1 antibody.
ing human CEA and mGM-CSF was used as a DNA vaccine
Accordingly, the 3H1 polynucleotides can be inserted in live
to immunize C57BL/6 mice. Mice immunized with this
or attenuated viruses (vaccinia virus) or viral vectors, such as
plasmid showed both CEA-specific anti-tumor cellular and
adenovirus, adeno-associated viruses (AAV) and SV40,
humoral responses and were better protected against a chal-
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,
HLA-A2.1/Kb mice immunized with DCs pulsed by CEA-
Similarly, Losman et al. developed a rat monoclonal anti-
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
MC38-CEA2 cells). Mice immunized with Tat-CEA-pulsed
soluble immunogenic carrier protein, optionally in combina-
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-
A fusion protein comprising a nucleotide sequence en-
22392 patent application provided modified versions, which
coding 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
Similarly, Kim et al. pulsed DCs with an mRNA encod-
for the human CEA that were immunized with this fusion
ing a fusion protein composed of TAT-PTD, calreticulin
protein showed stronger anti-CEA antibody responses to
(CRT) and a human truncated CEA (CRT-TAT-(cid:1)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-(cid:1)CEA. After immunization,
the capacity of anti-Id antibody vaccines to break peripheral
enhanced CEA-specific CD4+ and CD8+ T cell responses
tolerance against TAAs [51].
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
with the CEA protein. The double loading of DCs ensures
against CEA in BALB/c mice [52].
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 con-
firmed 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
demonstrated that the combined therapy using a DCs-based
protection against tumor growth was achieved when
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
useful for promoting adenoviral transduction and the matura-
that vaccination with DCs transduced with rAdV encoding
tion 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
Recombinant Vector-based Vaccines
mice). Vaccination with AdV-CEA/DCs promoted an im-
provement in the proliferation of antigen-specific T cells and One strategy to overcome the tolerance to CEA and to
the number of IFN-(cid:1) 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
a) CEA-viral Vectors
patent describes an antitumor vaccine and pharmaceutical
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 sig-
nificant 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
other pathologies in different mouse models bearing trans-
The same authors also patented a composition containing
genic 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 intratu-
moral boosting vaccination with rF-CEA/TRICOM plus rF-
McLaughlin et al. demonstrated that IL-2 administration
IL-2 and rF-GM-CSF was confirmed by Kudo-Saito et al.,
enhanced the therapeutic effects of rV-CEA in C57BL/6
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
crease in the survival of mice bearing multiple CEA+ intes-
molecules is represented by the work of Aarts et al., which
tinal 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-(cid:1) pro- with vaccine therapy remained tumor-free for a long period
duction, which allowed mice to counteract tumor progression of time [83].