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

in the generation of an antitumor response rendering the
tumor cells immunogenic; nontransfected tumors are not
rejected by the animal and grow causing its death (Tepper
et al, 1989; Fearon et al, 1990).
Ex vivo gene therapy trials using cytokine gene
transfer (see below) circumvent the problem of toxicity of
IL-2 administration; for non-gene transfer therapies, white
blood cells drawn from patients are fractionated, cultured,
stimulated with IL-2 or other cytokines, and reintroduced
in much higher numbers into the blood of the patient.
C. Cancer immunotherapy with cytokine
genes
The combination of immunotherapy with conventional
treatments such as radio- and chemotherapy may be
necessary to eradicate minimal residual disease. Advanced
therapies involve the transfection of lymphocytes in
culture with cytokine genes followed by selection of the
successfully transfected cells with a selectable marker
such as the bacterial neomycin-resistance gene (Cassileth
et al, 1995). Numerous phase I clinical trials employing
either syngeneic genetically modified or allogenic tumor
vaccines are in progress (see immunotherapy in Appendix
1, page 159-172). The development of tumor cells
transduced with cytokine genes and their exploitation as
tumor vaccines in patients with cancer is a very promising
field (reviewed by Jaffee and Pardoll, 1997).
Cytokine genes used for cancer immunotherapy
include those of IL-2, IL-4, IL-7, IL-12, IFNs, GM-CSF,
TNF-α in combination with genes encoding costimulatory
molecules, such as B7-I. The major goal of the
use of immunostimulatory cytokines is the activation of
tumour-specific T lymphocytes capable of rejecting
tumour cells from patients with low tumour burden or to
protect patients from a recurrence of the disease. As
distant metastasis is the major cause for therapeutic
failures in clinical oncology, treatment of patients having a
low tumor volume with immunotherapy could protect the
patient from recurrence of disease. Treatment of rodent
tumor models with little or no intrinsic immunogenicity
with this approach resulted in regression of preexisting
tumors and cure of the animals from their disease;
furthermore, in some instances cured animals had retained
immunological memory and resisted a second challenge
with the parental tumor cells (reviewed by Gilboa, 1996;
Mackensen et al, 1997).
The transduction of the tumor cells of the patient with
cytokine genes ex vivo and the development of tumor
vaccines depends on the establishment of primary cell
culture from the solid tumor. Although malignant
melanomas are easy to culture, it is difficult to establish
cell lines from most other primary human tumors using
convenient methods; primary tumor cultures are being
used (i) for the transduction of autologous cells from the
Boulikas: An overview on gene therapy
46
cancer patient with cytokine genes to develop cancer
vaccines after intradermal implantation to the patient; (ii)
for characterization of tumor-specific cytotoxic T
lymphocytes in order to identify specific antigens on the
human primary culture; (iii) for extensive phenotypic
characterization of the tumor in cell culture. The Cre/LoxP
system (see recombinases in gene therapy) has been used
to facilitate the establishment of primary cell lines from
human tumors (Li et al, 1997).
Human gene therapy protocols 3 and 10 (Appendix 1)
use immunization of cancer patients with autologous
cancer cells transduced with the gene for tumor necrosis
factor (TNF).
D. Cancer immunotherapy with the IL-2
gene
Active immunization with pancreatic tumor cells
genetically engineered to secrete IL-2 were shown to
inhibit pancreatic tumor growth in vivo; this was shown
using a poorly immunogenic subcutaneous model of
murine ductal pancreatic cancer by implanting tumor
Panc02 cells in C57BL/6 mice; whereas 90% of animals
vaccinated with irradiated parental Panc02 and
subsequently challenged with parental Panc02 cells
developed tumors by 48 days only 40% of animals
vaccinated with irradiated Panc02 cells engineered to
secrete IL-2 and challenged with parental Panc02 cells
developed tumors by 48 days (Clary et al, 1997).
According to a RAC-approved clinical protocol the
gene for human interleukin-2 (IL-2) was transduced into a
cell line established from the neoplastic cells of a patient
with malignant melanoma; this procedure established an
IL-2-secreting cell line with integration of the IL-2 gene
into genomic DNA. The IL-2-secreting cells were
irradiated, in a manner sufficient to inactivate 100% of the
cells but insufficient to completely inhibit IL-2 synthesis,
and administered to 12 patients with metastatic malignant
melanoma in a Phase I toxicity study. These cells have the
capacity to induce an antimelanoma response as shown in
animal studies (Das Gupta et al, 1997).
A significant number of RAC-approved clinical
protocols use IL-2 cDNA transfer. These include protocols
11, 16, 19, 20, 46, 48, 50, 61, 71, 102, and 135, in
Appendix 1 and protocols 190, 197, 198, 200, 204, 211,
213, 215, and 219 using cationic lipids for gene transfer
(Table 4 in Martin and Boulikas, 1998, this volume, pages
203-206).
E. Cancer immunotherapy with the IL-3
gene
IL-3 was found to enhance the development of
cytotoxic T lymphocytes; during antitumor response,
macrophages could ingest whole tumor cells, cell