01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

ecombinant vaccinia virus expressing the gene for murine
GM-CSF injected to solid melanoma tumors twice weekly
for 3 weeks; this injection regimen resulted in growth
inhibition of the subcutaneous tumor and enhanced the
survival of the animals (Ju et al, 1997).
A recent effort has been toward potentiation of Tlymphocyte-mediated
antitumor effects. T-lymphocyte
response incapacitation in the murine renal cancer model
could arise from an impairment of critical nuclear
transcription factors. A vaccine-oriented gene therapy
approach used T cells and antigen-presenting dendritic
cells which were recruited through the use of antigen,
chemokines and GM-CSF and further potentiated by
fibroblasts expressing IL-2, IL-4, IL-7, or IL-12; the goal
of this approach was to optimize MHC class I- and class
II-dependent pathways for induction of T-lymphocytemediated
responses to cancer in animal models (Wiltrout
et al, 1995).
Chen et al (1996) found that adenoviral delivery of a
combination of HSV-tk, mouse IL-2, and mouse GM-CSF
is much more effective for the treatment of metastatic
colon carcinoma in the mouse liver than HSV-tk alone or
HSV-tk combined only with IL-2; a fraction of the
animals developed long-term antitumor immunity and
survived for more than 4 months without tumor recurrence
in the three gene combination regimen; thus, local
expression of GM-CSF in the hepatic tumors and
prolonged IL-2 expression were necessary to generate
persistent antitumor immunity.
A gene gun device was used to accelerate and
introduce gold particles coated with GM-CSF cDNA
plasmids into mouse and human tumor cells. Transfected
and irradiated murine B16 melanoma cells produced about
100 ng/ml murine GM-CSF/million cells per 24 hr in vitro
for at least 10 days. Toward development of a tumor
vaccine, irradiated B16 tumor cells expressing murine
GM-CSF cDNA were then injected into mice. Subsequent
challenge of these mice with nonirradiated, nontransfected
B16 tumor cells showed that 58% of the animals were
protected from the tumor by the prior vaccine treatment
compared to only 2% of control animals inoculated with
irradiated B16 cells transfected with the luciferase gene
(Mahvi et al, 1996).
Human tumor tissue transfected within 4 hr of surgery
produced significant levels of transgenic human GM-CSF
protein in vitro. Human GM-CSF was readily detectable in
serum and at the injection site following subcutaneous
implantation of these transfected tumor cells into nude
mice (Mahvi et al, 1996).
The autocrine secretion of GM-CSF by transduced
tumor cells was found to serve as an effective immune
adjuvant in the host response to a weakly immunogenic
murine mammary carcinoma tumor: transfer of activated
lymph node cells derived from mice inoculated with GM-
CSF-secreting (240 ng/million cells/24 hours) murine
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mammary carcinoma cells resulted in the prolonged
survival of animals with macroscopic metastatic disease;
this was not evident utilizing lymph node cells from mice
inoculated with wild-type tumor (Aruga et al, 1997).
3. Clinical trials
Autologous cells (sensitized T cells) geneticallymodified
to secrete GM-CSF have been used for adoptive
immunotherapy on humans. GM-CSF has been used for
the treatment of advanced melanoma or renal cell cancers
(Chang et al, 1996). The steps included retrieval of tumor
from the patient for use as a vaccine; the tumor cell line
was transduced with a retroviral/GM-CSF vector; cells
were reintroduced into the patient (tumor vaccination).
Removal of draining lymph nodes after 7-10 days and
activation of lymph node cells with a monoclonal antibody
directed against CD3 and expansion of the cell population
with IL-2 gave anti-CD3 + /IL2-activated cells which were
exquisitely tumor-specific and mediated the regression of
established tumors in animal models (Figure 18).
According to a phase I clinical trial cancer patients are
intradermally vaccinated with lethally-irradiated tumor
cells that have been transfected by particle-mediated gene
transfer with gold particles coated with human GM-CSF
plasmid DNA; this is based on preclinical studies showing
that vaccination of mice with irradiated, GM-CSFtransfected
melanoma cells provided protection from
subsequent challenges with non-irradiated, non-transfected
tumor cells. Human tumor immunotherapy studies in
course use patients' fresh specimens of melanoma or renal
carcinoma; cells are dissociated, lethally-irradiated and
transfected with GM-CSF plasmid DNA-coated gold
particles resulting in the subsequent production of
biologically active GM-CSF protein by the patient’s cells.
Patient’s cells are used intradermally as a vaccine to elicit
anti-tumor immune responses. Surgical excision of the
vaccination sites will assess GM-CSF production and
infiltration of immune effector cells; patients are being
subjected to an intradermal injection in their opposite
extremity of 5 million irradiated cryopreserved tumor cells
taken from the patient at the time of vaccine preparation to
asses immune reactions (DTH testing); if a positive
reaction is noted on day 28 the DTH site will be surgically
removed (Mahvi et al, 1997).