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Leading article
Can immunotherapy by gene transfer tip the balance against
colorectal cancer?
Summary
Gene therapy, in particular the transfer of genes encoding
immunostimulatory molecules (cytokines and costi-
mulatory molecules) as well as selectively cytotoxic
enzymes and DNA vaccination, has the potential of
enhancing cell mediated immune responses against
tumours including those of colorectal origin. Genes can be
transferred using viral vectors either to cultured tumour
cells in vitro that can be returned to the patient as a “can-
cer vaccine”, or directly to tumour cells in vivo.
Vaccination with DNA constructs expressing specific
tumour antigens characteristic of colorectal neoplasia can
trigger immune recognition and destruction of tumour
cells. The aim is to tip the balance from protumour to
antitumour mechanisms by generating a local immune
response and systemic antitumour immune memory to
destroy metastases. Studies in murine models, combined
with human studies, show that such approaches could
become an adjunct to current treatments for human
colorectal cancer in the near future.
Introduction
Colorectal cancer comprises 10–15% of deaths from
cancer in industrial nations, second only to lung cancer.1
Survival rates (40% >5 years) have remained stable over
the past 20 years and so a number of treatments to supple-
ment surgical resection and chemotherapy are under
investigation, including enhancement of the immune
response. This article considers gene therapy, in particular
the transfer of immunomodulatory genes and selectively
cytotoxic enzymes to tumour cells as well as DNA vaccina-
tion, as a means of enhancing cell mediated immunity spe-
cifically for the treatment of colorectal cancer.
The current model for colorectal tumorigenesis postu-
lates a multi-stage progression involving an accumulation
of gene mutations (APC, K-ras, p53, DNA mismatch
repair genes), alterations in gene expression (c-myc,
MHC) and chromosome losses, during which regulation
of cell growth is disrupted.2Dietary and inherited genetic
factors predispose to such changes. The majority of deaths
from colorectal cancer follow tumour metastasis to the
liver and treatment must be aimed at controlling local
regrowth after resection and distant metastases. Cell
mediated immunity (mainly CD8+ cytotoxic T lym-
phocytes (CTL)) is potentially the most eVective arm of
the immune response as CTL can recognise epitopes
processed and presented from any protein synthesised
within the tumour cell and can kill the cell specifically and
also anamnestically (memory cells). Cytokines from
CD4+ helper T cells (Th) are also required to activate not
only CTL, but also natural killer (NK) cells and antigen
presenting cells and other inflammatory cells at the
tumour site. Although lessons can be learned from gene
therapy approaches against other tumours, mainly
melanoma,3colorectal cancers have characteristic features
which require separate consideration.
The immune response to colorectal tumours and
reasons for its failure
The emergence of a tumour may be the result of an inad-
equate immune response on two fronts: poor or lack of
immunogenicity of the tumour cells and low eYciency of
the immune response against the tumour. However, colo-
rectal tumours do not develop more frequently in
immunodeficient individuals, unlike some other
tumours—for example, lymphomas in patients with AIDS,
skin tumours in transplant recipients. This suggests that
the tumour itself has immunomodulatory or immuno-
evasive, or both, properties.
Tumour cells often fail to present antigen due to the total
loss (in around 20% of colorectal neoplasia) or reduction
in expression of MHC class I molecules.45 Mutations in
peptide transporting molecules (TAP) may also aVect
presentation of T cell epitopes.6The genetic changes
occurring during tumour development frequently lead to
the expression of oncogenic and neo-antigens (tumour
specific) or aberrant expression of normal or fetal antigens,
which are potential targets for immune attack of the cancer
cells. Antigens recognised by T cells in colorectal cancer
include mutated p21 ras78 cell surface associated mucin9
and an annexin-like molecule.10 However, for an eVective
antitumour response T cell specificities may need to be
directed towards subdominant or cryptic epitopes of
unmutated self molecules as dominant epitopes may have
induced thymic depletion or peripheral anergy of epitope
specific T cells.11 A precedent for this is seen in melanoma
where a number of self antigens are associated with protec-
tive immunity to tumours.12
The total or partial loss of MHC class I molecules means
that tumour antigens may not be presented to CTL if a
particular MHC class I allele is required for peptide pres-
entation. This provides a selective advantage for the
tumour cells and a problem for the immune response. In
addition, the presence of MHC class I alleles can inhibit
Leading articles express the views of the author and not those of the editor and editorial board.
Gut 1998;43:445–449 445
the non-specific cytotoxic activities of NK cells. Thus, cer-
tain phenotypes of MHC expression can render the
tumour cells non-susceptible to direct cellular cytotoxicity
by CTL or NK cells. Loss of polarisation at the luminal
membrane of epithelial tumour cells gives rise to aberrant
expression of mucin molecules (such as MUC-1).13
Expression of mucin all over the cell membrane can mask
surface immunoregulatory molecules and inhibit interac-
tion between tumour and immune cells.14 However,
changes in mucin glycosylation may make the mucin a tar-
get for CTL activity and Th cell recognition without MHC
restriction due to its repeating and possible TcR
cross-linking properties.15 An additional paradox in the
importance of mucin is the fact that MUC-1 expression on
adenocarcinomas (or shed from them) can cause apoptosis
of T cells,16 which is another way in which the tumour may
evade the immune response. A number of tumour infiltrat-
ing lymphocyte (TIL) populations have been identified in
colorectal tumours, which may have an association with
increased patient survival.17 18 Indeed, TIL cultured in vitro
and adoptively returned to patients have resulted in varying
degrees of protection in other cancers.12 NK cells are par-
ticularly abundant in colorectal TIL19 as are CD4+ T cells
which outnumber CD8+.20 21 Colorectal tumour infiltrat-
ing T cells with a limited repertoire of T cell receptor vari-
able regions suggests tumour specific clonal expansion22 23
as does the possession of activation markers.24 T cells with
the ãä T cell receptor, which have been identified in
neoplastic as well as in healthy intestinal mucosa,25 have
specific killing activity towards epithelial-derived tumours
in a non-MHC restricted manner26 and may provide
another antitumour mechanism. Furthermore, both ãä T
cells,27 and NK cells28 seem to recognise heat shock
proteins (hsp), molecules which are constitutively ex-
pressed by colorectal neoplasia29 (S Todryk, unpublished
data). The upregulation of hsp by heating30 or gene
transfer30a could therefore be another means of improving
immune recognition of colorectal cancer.
Cell mediated immunity tends to be down regulated in
environments such as the gut in order to minimise damage
caused by excessive inflammation in response to the
barrage of antigens encountered. Indeed, colon adenomas
and carcinomas produce transforming growth factor
(TGF) â31 32 and interleukin (IL) 10,33 cytokines known to
suppress cell mediated responses, an eVect that may be
more pronounced within larger, established tumours.
Secretion by colorectal tumours of factors such as leukae-
mia inhibitory factor and prostaglandins may also have
immunosuppressive eVects.34 35 This could, in part, explain
why these tumours tend to develop and persist, despite the
presence of TIL. This suppression may also occur in gut
associated lymphoid tissue, mediated by T cells.36 Recent
evidence, however, has shown that colorectal tumour cells
secrete IL-7, a cytokine that can cause TIL to proliferate,
secrete tumour necrosis factor (TNF) áand lyse
autologous tumour cells.37
The absence of costimulation (e.g. by helper cytokines or
B7 binding) during recognition of tumour cells by T cells
results in anergy of tumour specific T cells,38 rendering
them ineVective. Such anergy may need to be reversed in
immunotherapy. In humans with colorectal cancer the
functional suppression of T cells in the TIL and periphery
seems to coincide with alterations in the T cell receptor
signal transduction mechanism,39 40 but these may be
reversible by cytokines such as IL-2.41 Finally, colorectal
tumours express not only functional Fas ligand, which can
induce apoptosis in tumour infiltrating T cells bearing Fas,
but also Fas itself, which although expressed at lower levels
than in normal colon epithelium may make the tumour
cells susceptible to apoptosis.42 43
In conclusion, the fact that it is possible to detect cellu-
lar immune responses specific for colorectal tumours in
vitro,44–48 albeit at low levels, suggests that the immune
defect could be reversed in vivo by immunotherapy.
Gene transfer mediated immunotherapy of
colorectal cancer
IMMUNOSTIMULATORY GENES
Immunostimulatory gene transfer is a potentially powerful
therapeutic approach for treating colorectal cancer that
aims to mobilise the immune response to recognise and
destroy tumour cells (box). Gene transfer therapy usually
involves the resection of tumour and then infection in vitro
of tumour cells with retro-, adeno- or herpes viruses49 con-
taining genes for cytokines and/or costimulatory mol-
ecules. This is followed by reinjection of the irradiated or
unirradiated cells as a “cancer vaccine”. When cytokine
genes are transferred, tumour cells will secrete the cytokine
and stimulate immune responses and inflammation by a
local paracrine eVect. When genes for costimulatory
molecules (e.g. B7) are transferred the molecule will be
expressed on the tumour cell surface and stimulate
lymphocytes by direct contact. Local production of
cytokines also avoids toxic eVects of systemic cytokine
administration. Together with the elimination of the
inoculating tumour cells, this approach aims to elicit
systemic immune memory and protection against second-
ary contact with parental tumour (subcutaneous or in the
liver in the mouse model), which represents tumour
regrowth and metastasis in humans. A number of
transferred cytokines have shown varying degrees of
protection in tumour models,50 with IL-2,51 GM-CSF52–54
and IL-1255 56 being most eVective and consistent at induc-
ing protective immunity in murine colorectal tumour
models. We have found IL-12 and B7.1 to be a
combination that elicits the greatest degree of protection54
and IL-12 can also give rise to CTL that successfully treat
colorectal tumour “metastases” in the lung.57 The local
release of these cytokines induces a cell mediated Th type
1 response (IL-12) or the stimulation of dendritic cell pre-
cursors (GM-CSF), which take up tumour antigens,
migrate to the lymph nodes and prime T cells giving rise to
eVector and memory T cells.58 CTL and/or NK cells medi-
ate the tumour rejection in these models, with51 or
without55 T cell help. The inflammatory environment cre-
ated by the transferred cytokines also enhances the expres-
sion (together with MHC molecules) and recognition of
less dominant self-antigen T cell epitopes which can then
become targets for CTL.11 In comparison to such murine
models, humans may have carried their tumours for long
periods prior to gene therapy, and both priming of naive
cells and reversal of anergy will be required. In addition,
selective pressure over many years will have caused the
tumour to adapt to, and evade, the immune response. In
human studies, the transfer of the B7 costimulatory
molecule to colorectal tumour cell lines did not cause acti-
vation of T cells in vitro,59 whereas the transfer of IL-2
Aims of immunostimulatory gene transfer
therapy against colorectal cancer
+Induce an immunostimulatory environment in the
vicinity of the tumour/vaccine
+Induce direct or cross-priming of cytotoxic and
helper T cells against tumour antigens
+Overcome immunosuppression and/or T cell anergy
+Generate immune memory against tumour regrowth
and metastasis
446 Todryk, Chong, Pandha, et al
stimulated NK cells in vitro but not tumour specific CTL.60
In human melanoma, exogenous IL-12 and IL-2 in
combination, but not B7, yielded the best in vitro CTL
responses.61
In vivo infection of tumour cells with tumour targeting
viruses (systemic or local administration) may also be fea-
sible in gene therapy62 and avoids in vitro cell manipulation
for each patient, and the production of a “personal”
vaccine. Liver metastases could—for example, be targeted
by perfusion of the liver with viruses via the hepatic portal
vein or by intratumoural injection. Carcinoembryonic
antigen (CEA) expressing tumours could be targeted by
engineering proteins within the viral envelopes that bind
specifically to surface CEA, or by incorporating the CEA
promotor.63 This in vivo gene delivery approach should
aVect the growth of the targeted tumour and elicit protec-
tive immunity against spread of the tumour. Alternatively,
the possibility exists of using allogeneic tumour cells, with
antigens in common with the patient’s tumour, which will
be rapidly destroyed and these antigens released, resulting
in T cell cross-priming against the antigen. Established
tumours, or tumour cells ex vivo, can also be made alloge-
neic by transfer of allo-MHC genes.64 In addition to
tumour cell modification, transfer of the gene for TNF-áto
TIL from melanomas has been achieved,65 and may
provide another means of enhancing cell mediated immu-
nity against colorectal neoplasia using gene therapy.
SUICIDE GENES
Another form of gene therapy involves the in vitro
(followed by injection) or in vivo infection of tumour cells
with viruses carrying “suicide” genes which encode
enzymes (e.g. herpes simplex virus thymidine kinase (tk)
and Escherichia coli cytosine deaminase (CD)) that convert
prodrugs (ganciclovir and 5-fluorocytosine, respectively)
into toxic forms that kill the tumour cells in vivo. This
inflammatory process rapidly releases antigens that stimu-
late memory immune responses resulting in the killing of
parental tumour cells distal from the initial tumours (e.g.
metastases).62 66 This approach has been successful at
reducing growth of a tumour challenge in a mouse
colorectal tumour model67 (and our unpublished data).
Moreover, cotransfection of tk with GM-CSF, adminis-
tered in adenovirus in vivo, was able to increase survival of
mice with liver metastases.68 The use of suicide genes may
also reduce the need for tumour cell irradiation, which
could adversely aVect the vaccine’s eYciency.69 The
tumour cells would be killed when the prodrug is adminis-
tered. In a comparison between tk and CD gene transfer,
CD was more eVective than tk at killing a human colorec-
tal tumour line in nude mice using in vitro,70 or in vivo
delivery (adenovirus).71 However, colorectal tumour lines
passaged over many years may not provide the most accu-
rate model for colorectal cancer therapy. For this reason we
are currently studying tumour cells freshly isolated from
patients.
DNA VACCINATION
The administration of genes encoding tumour associated
antigens provides another potential route of immuno-
therapy against colorectal cancer. Antigen encoding
plasmid DNA can be given in its naked form by intrader-
mal or intramuscular routes, and by injection or “gene
gun”. Alternatively, vehicles for DNA vaccination include
liposomes, viral vectors and protein carriers. The pro-
longed antigen expression that is obtained can induce CTL
and Th responses.72 Even though relatively few antigen
specific T cell responses have been identified for colorectal
tumours, as previously mentioned, there are a number of
candidate proteins that could be exploited as DNA
vaccines (table 1). Potential tumour specific antigens are
those expressed uniquely by the tumour, or in greater
abundance than normal tissue. In addition, as T cells rec-
ognise peptide epitopes of around eight to 20 amino acids,
MHC restriction of peptide recognition by the heterog-
enous human population may necessitate the use of larger
antigenic fragments that encompass many epitopes.
Mutations in oncogenes may be single amino acid
changes, as at codons 12, 13, and 61 in p21 ras. These
mutations disrupt normal ras signalling function and are
not expressed in normal tissue. Human CTL that
recognise a single ras mutation at residue 13 and are capa-
ble of killing tumour cells harbouring the same mutation
have been isolated from a patient with colon carcinoma.73
Peptides from this region of ras also bind MHC class II
molecules with high promiscuity,74 which is a desirable
attribute of a vaccine. Mutations in the p53 tumour
suppressor protein can give rise to multiple amino acid
substitutions. Such changes mean that cell growth is
unchecked and further gene mutations and chromosomal
rearrangements can accumulate. Murine CTL have been
raised to p53 mutated at codon 135.75 Carcinoembryonic
antigen is a glycosylated single-chain peptide overex-
pressed in carcinomas of the colon, breast, stomach,
pancreas, and lung. Promising CEA DNA vaccination
studies in mice76 77 using naked DNA or a vaccinia virus
vector are beginning to translate to human studies where
CTL generated by vaccinia-CEA immunisation could lyse
CEA+ tumour cells.78 Polymorphic epithelial mucin
encoded by the MUC-1 gene is overexpressed in a number
of adenocarcinomas. The mucin expression is no longer
only associated with the apical surface of ductal epithelial
cells and aberrant mucin glycosylation on tumour cells
results in exposure of the polypeptide core and unmasking
of otherwise cryptic epitopes. Immunisation with MUC-1
DNA, again naked or in vaccinia virus, has shown protec-
tion against tumour growth in mice.79 80 Although most
frequently associated with melanoma, the MAGE family of
genes has also been found in colorectal neoplasia81 and so
represent another potential candidate for DNA vaccina-
tion. Finally, mutational frameshifts such as those associ-
ated with APC gene expression can result in a stretch of
unique protein sequence containing potential T cell
epitopes.82
Recently, a potentially eVective route of DNA vaccina-
tion has emerged in which dendritic cells are pulsed or
transduced with tumour antigen encoding DNA and can
eYciently prime T cells.83 84 In light of the identification of
a number of tumour-regression antigens in melanoma and
Table 1 Candidate antigens for DNA vaccination against colorectal cancer
Antigen Class of antigen T cell responses Antitumour eVect/association
p21 ras Mutated oncogene product Human CTL, Th1 No
p53 Mutated tumour suppressor Murine CTL No
CEA Embryonic gene product Human CTL In mice
MUC-1 Epithelial mucin Non-MHC CTL In mice
MAGE Melanoma associated antigen Human CTL in melanoma In melanoma
GA733 Surface molecule Murine CTL, Th In mice88
Annexin-like molecules Placental/structural protein Th No
Treatment of colorectal cancer by gene transfer 447
other tumours, a requirement exists for the identification of
further tumour antigens in colorectal cancer.
Conclusion and future prospects
A number of cells of the immune system may be manipu-
lated in the gene therapy of colorectal cancer in order to tip
the balance from protumour to antitumour mechanisms
(fig 1). One of the challenges is to stimulate an eVective
immune response towards the various tumour phenotypes
and locations by transferring genes encoding the appropri-
ate immunostimulatory or cytotoxic molecules, or by
immunising with the appropriate tumour antigen encoding
DNA. EVective immune responses to colorectal neoplasia
that express or fail to express MHC class I molecules—for
example, may require diVerent immunostimulatory mol-
ecules to activate diVerent eVector cells.85 Tumour burden,
and in particular the size of the tumour, may very much
determine the success of such therapies as an immunosup-
pressive environment may be created and the tumour may
simply be proliferating too rapidly for the immune system
to contain. Previous studies immunising patients with
colorectal cancer with autologous tumour cells and bacillus
Calmette-Guerin (BCG) have shown some improvements
in survival rates86 and current gene transfer trials involve
transfer of allo-MHC molecules (HLA-B7)87 and cytosine
deaminase to colorectal tumours.3Genes encoding IL-12,
GM-CSF, B7, cytosine deaminase, and thymidine kinase
have shown therapeutic eYcacy in murine models of
colorectal cancer. DNA vaccination studies in murine
models that are currently translating to human studies
include CEA.
Colorectal cancer is amenable to gene therapy as
patients can be returned to a state of minimal residual dis-
ease following resection of the primary tumour. Latent
micrometastses will be a more controllable target. To put
these principles into practice we are currently working
towards clinical trials comprising in vitro transfection of
colorectal tumour cells with adenovirus encoding genes for
tk and GM-CSF, followed by reinjection of the cells as a
“vaccine”.89 These gene therapy approaches have the
potential to be useful adjuvants to conventional treatments
with potential advantages of being physiologically less toxic
and providing systemic vigilance against tumour regrowth
and metastasis.
This work is supported by the Lewis Family Charitable Trust (SMT) and the
Imperial Cancer Research Fund.
S M TODRYK
H CHONG
R G VILE
Laboratory of Molecular Therapy,
Imperial Cancer Research Fund Molecular Oncology Unit,
Imperial College School of Medicine,
Hammersmith Hospital, London W12 0NN, UK
H PANDHA
N R LEMOINE
Laboratory of Molecular Pathology,
Imperial Cancer Research Fund Molecular Oncology Unit,
Imperial College School of Medicine,
Hammersmith Hospital, London W12 0NN, UK
Correspondence to: Dr Stephen Todryk (email: s.todryk@icrf.icnet.uk).
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Figure 1 Tipping the balance in colorectal cancer.A number of
protumour mechanisms outweigh antitumour mechanisms and allow
colorectal tumours to survive and proliferate. Gene therapy with cytokine,
immunostimulatory or suicide (prodrug activating) gene transfer to
tumour cells, or DNA vaccination, aims to tip the balance towards
antitumour mechanisms and tumour rejection by enhancing antitumour
cellular immune responses. Correction of immune deficiencies associated
with the tumour could work in synergy with enhancement of antitumour
immunity and many of the genes shown could apply to both sides of the
balance.
Protumour mechanisms
Loss or partial loss of MHC molecules
FasL mediated apoptosis of T cells
MUC-1 mediated apoptosis of T cells
Immunosuppressive cytokines
No tumour antigen release
Antitumour mechanisms
MHC restricted CTL
MHC non-restricted NK cells
Fas mediated apoptosis of tumour cells
γδ T cell recognition of hsp
CTL recognition of MUC-1
Tumour antigen release and cross-priming
IL-12
GM-CSF
Prodrug activating
genes
DNA vaccines
IL-2
B7
Tumour
persistence
Gene therapy to counter
protumour factors
Gene therapy to increase
antitumour factors
Tumour
rejection
448 Todryk, Chong, Pandha, et al
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Treatment of colorectal cancer by gene transfer 449
