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

A. The molecular basis of cancer
immunotherapy
Many human tumors are nonimmunogenic or weakly
immunogenic. The immune system, evolved to rid the
body of unwanted intruders, could be instructed and
reinforced to eliminate cancer cells. Increasing the
immunogenicity of tumors by causing local cytokine
production or by increase in the expression in MHC
antigen can lead to local antitumor effect (Tepper et al,
1989; Fearon et al, 1990). Indeed, immune surveillance is
of the major defense mechanisms against cancer;
immunosuppressed individuals are more prone to cancer
and nude mice, lacking immune response, are exploited in
the lab to elicit tumors after injection of tumorigenic cells,
a response that, in many cases, cannot be elicited in
normal mice.
Cells undergoing malignant transformation are
believed to be eliminated from the body by white blood
cells including natural killer cells (NK), lymphokineactivated
killer cells (LAK), cytotoxic T lymphocytes
(CTL), tumor-infiltrating lymphocytes (TIL), and
activated macrophages; since established cancers in the
human body may escape this potential defense mechanism
of immunologic surveillance, cancer patients have been
treated with IL-2 to stimulate their cellular immune
mechanisms to kill cancer cells; lengthy and complete
remissions, however, were at a low rate and complications
were encountered by the toxicity caused by the systemic
administration of IL-2 (Rosenberg, 1992).
Transfection of the IL-2 gene into human melanoma
cells increased cellular immune response (Uchiyama et al,
1993). This and similar approaches have established the
foundation of the ex vivo cancer immunotherapy by
transfer of autologous (cancer patient’s) cells after
transduction in vitro with cytokine genes (see below). The
ultimate goal is the activation of tumour-specific T
lymphocytes capable of rejecting tumour cells from
patients.
B. Cancer immunotherapy with tumor
infiltrating lymphocytes (TILs)
Ex vivo approaches in immunotherapy have been
aimed at isolating T cells directly from tumors (known as
tumor infiltrating lymphocytes or TILs), stimulate TILs to
proliferate in cell culture with IL-2 followed by their
reintroduction into the blood stream of advanced cancer
patients (Rosenberg et al, 1988). The adoptive transfer of
TILs was 50-100 times more potent than that of
lymphokine-activated killer (LAK) cells isolated from the
patient's tumors. Treatment of 20 patients with TILs after
their expansion in vitro, plus IL-2, resulted in objective
regression of metastatic melanomas in lungs, liver, bone,
skin, and subcutaneous sites which lasted for several
months (Rosenberg et al, 1988).
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TILs were also transfected in vitro with the bacterial
neomycin-resistance gene and were reintroduced into
patients with advanced cancer in order to follow their
persistence in blood circulation with PCR methods
(Aebersold et al, 1990). Such “gene marking” clinical
protocols using TILs are numbers 1, 3, 9. 13, 57, and 169
in Appendix 1, pages 159-172. Having shown safety in
the NeoR-modified TIL protocol, the gene for tumor
necrosis factor (TNF) was added to the vector for therapy
of malignant melanoma in advanced cancer patients; the
first patient began treatment in January 1991. TNF,
however, is effective as an anticancer agent in mice at 400
mg/kg body weight, but in humans, TNF is toxic at 8
mg/Kg and so far of no proven therapeutic value at this
low concentration (reviewed by Anderson, 1992). In a
similar approach, the TNF gene was replaced by the gene
of interleukin-2 (IL-2) in order to develop locally high
doses of IL-2 at the tumor site by immunization with TIL
cells from the patient producing systemic antitumor
immunity (Rosenberg et al, 1992).
TNF-α, (also IL-1β, IFN-γ, and vitamin D3) after
binding to their transmembrane receptors stimulate the
production of the second messager ceramide from
sphingomyelin in the plasma membrane by activating
sphingomyelinase; this results in a cascade of signal
transduction events that result in down regulation of c-myc
and induction of apoptosis, to terminal differentiation, or
to RB-mediated cell cycle arrest (see apoptosis further
below).
IL-2 stimulates the differentiation of precursor
lymphocytes into LAK cells and further stimulates LAK
cell proliferation; LAK cells are probably produced by
activation of NK cells or from activated T cells by IL-2.
Administration of IL-2 plus amplified LAK cells into mice
models led to marked regression of disseminated cancers
and leukemia. LAK cells are able to destroy tumor cells
that express only weakly histocompatibility antigens. IL-2,
however, has several pleiotropic effects: stimulation of B
cell proliferation; activation of HLA class II antigen
expression on endothelial cells, TILs, and melanoma cells;
and enhanced production and release of TNF-α, and IFN-γ
(see Cassileth et al, 1995 and the references cited therein).
However, the use of large numbers of adoptively
transferred, broadly cytotoxic LAK cells in combination
with IL-2 has been effective for only small subsets of
cancer patients (reviewed by Wiltrout et al, 1995).
TILs, which could potentially kill tumor cells, are
found in many tumors but remain suppressed or anergic;
this anergy may arise from the absence of lymphokines
which provide signals for TIL cell activation and
stimulation to proliferation although ligands may be bound
to the variable region of the T cell receptor; indeed,
nonimmunogenic tumors are rejected by syngeneic mice
upon transfection by IL-2 or IL-4 genes; IL-2 lymphokine
production by the tumor cells bypasses T helper function