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-Alfred E. Chang, M.D.,
Chief, Division of Surgical Oncology,
Director, Gene Therapy Program
Adoptive immunotherapy is traditionally defined as the passive
transfer of immunologically competent tumor-reactive cells
into the tumor-bearing host to, directly or indirectly, mediate
tumor regression. The feasibility of adoptive immunotherapy
of cancer is based on two fundamental observations derived
from extensive experimental animal studies. The first of these
observations is that tumor cells express unique antigens that
can elicit an immune response within the syngeneic host. The
other is that the immune rejection of established tumors can
be mediated by the adoptive transfer of appropriately sensitized
lymphoid cells. Recognizing these fundamental principles required
the establishment of animal models consisting of inbred strains
of rodents and syngeneic transplantable tumors to eliminate
the confounding influences of allograft transplantation immunity
observed in earlier studies of tumor rejection in non-inbred
animals. From these animal models, several principles have
been established. These principles are summarized in Table
Requirements for Clinical Therapy
As previously indicated, large numbers of immune cells are
required to mediate the regression of an established tumor.
However, unlike experimental animal systems, humans do not
have readily available genetically identical counterparts
to obtain immune cells. Therefore, tumor-reactive lymphoid
cells will have to be identified and isolated from the patient
with cancer. Furthermore, to generate sufficient quantities
of immune cells, in vitro methods of expanding these cells
while maintaining their immunological reactivities are required
to render clinical therapy feasible. These represent formidable
The requirements for successful adoptive immunotherapy in
humans is summarized in Table 2. The ability to retrieve tumor-sensitized
cells from the total pool of lymphoid cells available in the
patient is of foremost importance. Human cancers spontaneously
arise and may not be sufficiently immunogenic to allow the
isolation of immune T cells. There are three potential sources
to retrieve lymphoid cells for possible isolation of immune
cells in humans which consist of: 1) peripheral blood, 2)
lymph nodes or 3) tumor. The frequency of tumor-reactive lymphoid
cells in the peripheral blood is exceedingly low. Hence, we
and other investigators have been interested in isolating
immune T cells from either a growing tumor (a.k.a. Tumor-Infiltrating
Lymphocytes, TIL) or from lymph nodes draining sites of tumor
vaccination (a.k.a. Vaccine-Primed Lymph Node cells, VPLN).
We have developed methods to culture these T cells ex vivo
in large quantities for subsequent infusion for adoptive immunotherapy
in humans. We are currently performing several clinical trials
to evaluate these approaches, which are briefly summarized
in the next sections.
VPLN Cells Primed by Autologous Tumor plus BCG
In extensive animal studies, we have demonstrated that we
can sensitize T cells to a non-immunogenic tumor by vaccinating
the host with irradiated tumor cells mixed with a bacterial
adjuvant (1). These VPLN can be surgically retrieved and expanded
ex vivo using an antibody to T cells (i.e., anti-CD3) along
with Interleukin -2 (IL-2). These cells are highly effective
in mediating the regression of advanced tumors in mice. The
bacterial adjuvant is important to boost the immune
response to weak tumor antigens. We have translated these
findings into a clinical protocol.
In this protocol, patients are vaccinated with irradiated
tumor cells mixed with the bacterial agent, BCG. The tumor
cells have been previously retrieved from the patient and
should express all the tumor-associated antigens unique to
that individual patient. The vaccine is inoculated intradermally
and approximately one week later the VPLN cells are harvested
in a minor outpatient procedure. In preliminary studies we
have performed, the VPLN cells exhibit a significant degree
of specific tumor-reactivity as measured in in vitro assays.
For example, when these VPLN cells are exposed to autologous
tumor cells in vitro, they secrete significant quantities
of interferon-gamma, an important cytokine involved in the
tumor rejection response (2,3). By contrast, these VPLN cells
do not react to tumor cells from other patients (Figure 1).
The VPLN cells are expanded in large quantities ex vivo and
are subsequently transferred back to patients intravenously
along with the concomitant administration of IL-2. To date,
we have seen signif-icant responses in several patients with
renal cell cancer (Figure 2). We are currently conducting
these trials in patients with advanced renal cell cancers,
sarcomas, and head and neck cancers.
VPLN Cells Primed by Genetically Engineered Tumor
More recent approaches for making tumor cells more immunogenic
have been to genetically modify the cells to elaborate cytokines
or express foreign antigens. In our laboratory, we have found
that the engineering of tumor cells to secrete GM-CSF has
dramatically enhanced their immune properties as a vaccine
(4). Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)
is a cytokine that recruits and promotes the proliferation
of dendritic cells at sites where cytokine-secreting tumor
cells are inoculated. This is thought to be important since
dendritic cells are key to processing and presenting antigen
to T cells.
In a clinical trial, we are genetically modifying tumor cells
to secrete GM-CSF using a retroviral vector. These cells are
then irradiated and inoculated intradermally as a vaccine
to prime draining lymph nodes. One week later, the VPLN are
retrieved using a blue dye technique to identify the immediate
draining lymph nodes (Figure 3). The VPLN cells are expanded
in a similar fashion to what we described earlier and subsequently
transferred intravenously into patients. A patient with a
complete clinical response is illustrated in Figure 4. This
protocol is being performed in patients with stage IV melanoma.
TIL Derived from Genetically Modified Tumors
The University of Michigan has pioneered the applications
of direct gene transfer into tumors using non-viral vectors
(5). In patients with recurrent melanoma tumors, we have performed
intralesional injections of therapeutic genes complexed with
liposomes. The gene encodes for a foreign major histocompatibility
complex (MHC) class I protein, which is taken up by tumor
cells and causes them to express this foreign protein. We
hypothesize that the expression of the foreign protein by
the tumor cells induces a brisk inflammatory response within
the tumor leading to augmented immune responses to tumor-associated
antigens. In our analysis of this gene transfer approach,
we have compared the cytolytic activity of TIL retrieved from
the patients before and after gene injections and have found
enhanced TIL reactivity due to the injections (Figure 5).
In some patients, we have documented the regression of the
injected tumor nodules. In our current studies, we are taking
the TIL from patients after intralesional gene injections
and using them for adoptive immunotherapy of residual disease.
This study is for patients with stage IV melanoma.
In summary, the experimental observations that appropriately
activated lymphoid cells can mediate regression of established
tumor has led to the institution of clinical trials with encouraging
results. Despite this limited success, further elucidation
of the principles involved in sensitizing T cells to tumor
antigens will allow broader applications of this therapeutic
- Geiger JD, Wagner PD, Cameron MJ, Shu S, Chang AE. Generation
of T cells reactive to the poorly immunogenic B16-BL6 melanoma
with efficacy in the treatment of spontaneous metastases.
J Immunother. 13:153-165, 1993.
- Chang AE, Aruga A, Cameron MJ, Sondak VK, Normolle DP,
Fox BA, Shu S. Adoptive immunotherapy with vaccine-primed
lymph node cells secondarily activated with anti-CD3 and
Interleukin-2. J Clin Oncol. 15:796-807, 1997.
- Aruga A, Aruga E, Tanigawa K, Bishop DK, Sondak VK, Chang
AE. Type 1 versus type 2 cytokine release by Vß T
cell subpopulations determines in vivo antitumor reactivity.
J Immunol. 159:664-673, 1997.
- Arca MJ, Krauss JC, Aruga A, Cameron MJ, Shu S, Chang
AE. Therapeutic efficacy of T cells derived from lymph nodes
draining a poorly immunogenic tumor transduced to secrete
granulocyte-macrophage colony-stimulating factor. Cancer
Gene Ther. 3:39-47, 1996.
- Nabel GJ, Gordon D, Bishop DK, Nickoloff BJ, Yang Z-Y,
Aruga A, Cameron MJ, Nabel EG, Chang AE. Immune response
in human melanoma after transfer of an allogeneic class
I major histocompatibility complex gene with DNA-liposome
complexes. Proc Natl Acad Sci USA. 93:15388-15393, 1996.
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