Download Immune modulation as cancer treatment using gene therapy

BUMC Proceedings 1999;12:231-237
Immune modulation as cancer treatment using gene therapy
JOHN NEMUNAITIS, MD, AND JOSEPH A. KUHN, MD
From the Mary Crowley Medical Research Center, Dallas, Texas (Nemunaitis and Kuhn); US Oncology,
Dallas, Texas (Nemunaitis); and Baylor University Medical Center, Dallas, Texas (Nemunaitis and Kuhn).
Supported by a grant from Chiron Corporation, Emeryville, California.
Corresponding author: John Nemunaitis, MD, PRN Research, Inc., 3535 Worth Street, Collins Building, 5th
Floor, Dallas, Texas 75246.
Modulation of the immune system as an approach to attack cancer has
been explored for the past 50 years. Efficacy of this treatment has been
achieved in a limited number of patients. However, a major obstacle of
immune therapy is the toxicity related to the nonspecific nature of
immune activation. Efforts to improve the specificity of the immune
response have been investigated through the use of gene therapy.
Clinical trials involving gene therapy for melanoma, lung cancer, and
head and neck cancer have been conducted by US Oncology at the
Mary Crowley Medical Research Center–Baylor University Medical
Center. These studies represent one of the most active gene therapy
programs in the USA. Preliminary results have helped define the
mechanism of action, safety, and potential efficacy of immune
stimulatory treatment approaches in oncology.
odulation of the immune system to treat cancer has been tested extensively in
oncology. Cancers thought to be most sensitive to immunotherapy include melanoma, renal
cell carcinoma, colorectal cancer, and non–small cell lung cancer. Common immunotherapy
approaches have involved the use of interleukin (IL)-2 (1), interferon (IFN)-alpha-2b (2),
antibody therapy with muromonab-CD3 (3), lymphokine-activated killer cell infusions (4),
tumor-infiltrating lymphocyte infusions (5), dendritic cell infusions (6), peptide-stimulated
vaccine infusions (7), and various combinations of these. A limited number of successes
have been observed (approximately 20% in patients with metastatic melanoma). However,
toxicity related to nonspecific immune activation limits dosing and the effectiveness of
these approaches. Thus, more recently, a strategy of tumor-specific immune activation has
been explored involving gene therapy.
Key components of the immune system critical to understanding the therapeutic approaches
described in this review may require definition. Antigens are targets for immune response
that are located on the surface of malignant cells. Antigen-presenting cells (i.e., dendritic
cells) are cells that roam the body in search of foreign antigens. Antigenic peptides are
fragments of antigens that can be processed inside antigen-presenting cells and redisplayed
on the cell surface through linkage to major histocompatibility complex (MHC) molecules
that bind to antigen peptides for the purpose of cell surface display (Figure 1). T
lymphocytes contain receptors that recognize MHC/antigenic peptides. These cells become
activated upon recognition and binding. Lymphokines such as IL-1, IL-2, IFN, and GM-CSF
(granulocyte-macrophage colony-stimulating factor) are released by lymphocytes when
stimulated and induce proliferation and activation of other lymphocytes (B cells) and
monocytes. B lymphocytes recognize circulating antigens without MHC binding and
produce antigen-specific antibodies, which mediate antitumor effects through binding to
tumor antigens.
Some therapeutic approaches involve the following:
1.
2.
3.
4.
Improving expression (surface display) of antigens
Improving the number and sensitivity of antigen-presenting cells
Creating antigenic peptides in order to stimulate antigen-presenting cells (in vitro)
Altering MHC so that antigenic peptides can be exposed, thereby uncloaking
malignant cell identity to immune effector cells attempting to attack the cancer
Education of the immune system to recognize malignant cells intensifies the cellular
interaction and provides a focus for the immune attack. Other immunotherapy options
available today focus on the induction of cytokines or the addition of appropriate cytokines
to maximize anticancer effects. Malignant cells evade the immune system, in some cases, by
decreasing MHC levels and/or producing factors that inhibit antigen-presenting cell function
or T-cell or B-cell function.
One method of providing tumor antigen stimulation is to surgically harvest a tumor growing
in a patient and to modify the tumor ex vivo (in the laboratory) so that it will no longer grow
but still displays its surface antigens to stimulate the immune system when replaced in the
body. This is known as vaccine therapy. Tumor vaccines have been shown to be efficacious
in some clinical trials, primarily those involving melanoma, renal cell carcinoma, or
colorectal cancer (8–11).
The vaccine effect can be enhanced by several methods. Coincubation of the defective
antigenetic autologous tumor cells with bacillus Calmette-Gu?rin (a nonpathogenic bacterial
species) can enhance the inflammatory response to the vaccine, thereby improving the
antitumor systemic effect. Vaccine approaches can also use cancer cell lines that are
expected to have antigens similar to the patient's tumor. These cell lines are lysed following
incubation with oncolytic viruses and are then injected into the patient to stimulate an
antitumor response. Tumor vaccines have been shown to be effective in animal tumor
models.
The mechanism for the antitumor effect includes induction of antigen-specific antitumor
immunity mediated by CD4 and CD8 T cells. A comparison of vaccine approaches is shown
in Figure 2. Most recently, gene therapy has been used to increase antigen surface
expression in tumor cells via intratumoral injection rather than resection of the malignant
tissue for ex vivo processing.
Insertion of immune-modulating cytokine genes (such as IL-2, IL-4, gamma-IFN, tumor
necrosis factor [TNF]-alpha, M-CSF [macrophage colony-stimulating factor], or GM-CSF)
into tumor cells is another form of gene therapy designed to enhance an immune response
against implanted human tumors (12–26). Subcutaneous injection of cytokine-transduced
tumor cells in animal tumor models has been shown to induce prolonged tumor-specific
immune responses and improve survival from lethal tumor challenges. One of the earliest
studies showing such an effect involved injection of the TNF gene into murine sarcoma
tumors, which were then implanted subcutaneously into nude mice (27). Implantation of
tumor cells transduced with genes (neomycin) that do not affect immune responsiveness did
not change tumor growth. However, implantation of TNF-transduced tumor cells induced
significant regression of tumors compared with controls (Table 1). Tumor regression related
to the implantation of TNF-transduced sarcoma cells appeared to be mediated by CD4- and
CD8-positive lymphocytes. Data also revealed that implantation of identical sarcoma cells
not transduced with any genes following implantation of the TNF-transduced cells was
associated with regression of implanted secondary tumors, suggesting long-term systemic
antitumor activity. Implantation of tumor cells (i.e., breast cancer cells) not used as the
initial vaccine did not induce an immune response, thereby suggesting that the induced
systemic approach was specific against the tumor used as the gene-transduced vaccine.
Clinical testing would need to confirm that enhanced targeting of the immune system
against the cancer with gene therapy will increase immunogenicity and reduce systemic
toxicity. Defects that limit immune recognition of cancer are listed in Table 2. New
therapeutic approaches, including gene therapy, are designed to target those defects.
Results of the in vitro and in vivo studies described above were felt to be encouraging, thus
justifying development of clinical trials within our program initially using retroviral vector
gamma-IFN gene. The published results are summarized below.
Autologous Vaccination of Immune-Modulating Genes
Our first trial involving gene therapy to enhance antitumor immunity was performed in
1994 as part of the Baylor/PRN/Mary Crowley gene therapy program. Sixty-four patients
with advanced melanoma were entered into the trial in order to harvest autologous tumor
tissue and create a vaccine by introducing the gene for gamma-IFN (via a retroviral vector)
into the tumor cells (Figure 3) (28). Seventy surgical tumor specimens were processed from
58 evaluable patients. The median age of treated patients was 53 years (range, 17–83 years).
Of the 58 patients who underwent tumor resection, 12 tumor samples could be transduced
with the gamma-IFN gene. Adequate transgene expression (gamma-IFN protein),
production of functional gamma-IFN protein, and up-regulation of MHC-I and MHC-II
molecules on transduced tumor cells were confirmed in the 12 tumor cell lines.
The majority of patients were, however, unable to achieve autologous tumor cell line
expansion, generally because of overgrowth of fibroblasts, which were mixed in with the
malignant tissue at the biopsy. Additional difficulties with processing involved the time to
achieve the targeted minimum of 1 X 107 cells prior to transduction. The duration from
harvest of tumor tissue to release for clinical treatment ranged from 61 to 168 days. During
this time, several patients had disease progression despite standard care management and
were unable to participate in the treatment phase of the trial. However, 5 patients achieved
sufficient cell numbers and gamma-IFN expression to undergo treatment (subcutaneous
injection every 2 weeks) with the retroviral vector gamma-IFN gene vaccine. One patient
received 13 injections, 1 patient received 10 injections, and 3 patients received <=5 vaccine
injections. The patients receiving the fewest injections had a poor survival (35–72 days).
However, of the 2 patients who received 10 or 13 injections, 1 survived 885 days, and the
other remains alive without evidence of disease after >5 years. This patient had metastatic
disease to his liver, brain, and adrenal gland. All lesions were surgically resected creating a
minimum disease state, and the vaccine was administered every other week for 13
injections. Clinical toxicity to the gene-transduced vaccine was not observed in any of the 5
treated patients. Furthermore, no evidence of retroviral vector contamination was detected
in the released cell product or in patients following treatment.
Given the advanced stage of disease, it was difficult to evaluate efficacy. However, it is
highly unlikely that patients with brain and solid organ metastases survive more than 1 year,
although a patient in our study has survived 5 years without disease recurrence.
Nevertheless, spontaneous complete remission occurs at a 0.1% frequency in melanoma
(29), so additional trials were needed.
Researchers at Duke University have looked at retroviral gamma-IFN gene-transduced
autologous melanoma cell injection for treatment of metastatic melanoma, using an
approach identical to ours, by processing autologous harvested melanoma tissue and
transducing it with retroviral gamma-IFN gene (30). In our trial, all treated patients received
low-dose IL-2 (1.1 X 106 U) following vaccine injection, whereas in the Duke trial IL-2 was
not administered to patients. At Duke, 175 tumor samples were harvested. In 20 patients
sufficient gamma-IFN vaccine was produced for treatment. Gene expression and MHC upregulation were also confirmed. No toxicity was observed in the treated patients, and 4
patients achieved a minor response to vaccine. Thus, in both trials injection of retroviral
gamma-IFN gene-transduced autologous melanoma cells was well tolerated and antitumor
activity was observed. However, processing of the ex vivo vaccine severely limited the
number of patients able to be treated.
Immunotherapy with IL-2 gene-transfected melanoma cells has also been explored in one
phase I trial involving stage IV melanoma patients (31). Cell suspensions from surgically
harvested melanoma tumors were transfected with IL-2 gene and irradiated prior to
injection. Fifteen patients received injections of the vaccine. Observed toxicity was limited
to local erythema at the injection site and flulike symptoms in a few patients. Delayed-type
hypersensitivity reactions developed in 8 patients following injection; however, a 50%
tumor regression was not observed in any patients, although 3 patients with progressive
disease maintained stable disease for >=3 months.
The combination of systemic gamma-IFN and IL-2 in murine models has shown enhanced
antitumor activity compared with activity of either agent alone (32, 33). gamma-IFN
functions to enhance immunogenicity of the targets by activating nonspecific cytotoxic
effector cells (34). Specifically, gamma-IFN increases the expression of cell surface
molecules including MHC molecules and tumor-associated antigens (35). IL-2 enhances the
cytotoxic effect of gamma-IFN by providing a costimulatory signal that is necessary to
activate and expand cytotoxic T cells (34). However, toxicity related to the combination of
IL-2 and gamma-IFN administered systemically severely limits therapeutic effects observed
in animal models.
When the therapeutic effectiveness of the gamma-IFN–transduced tumor cell vaccine was
compared with an IL-2–transduced tumor cell vaccine in mice with established pulmonary
metastasis following infusion with B16 melanoma cells, both approaches were shown to
inhibit primary tumor establishment when vaccines were injected subcutaneously before
inoculation with unmodified tumor cells (14). However, mice treated with gamma-IFN–
secreting melanoma cells showed greater responsiveness and improved survival when
compared with mice treated with melanoma/IL-2–secreting cells. Combination of IL-2–
secreting cells and gamma-IFN–secreting tumor cells also showed no additional efficacy
compared with vaccination with either gene-transduced population alone.
SINGLE-COURSE INTRATUMORAL INJECTION OF GAMMA-IFN
RETROVIRAL VECTOR IN PATIENTS WITH METASTATICMELANOMA
In our program's second trial, 13 patients with metastatic melanoma were treated with
intratumoral injection of the retroviral gamma-IFN gene vector rather than removal of the
tumor and transduction of the gamma-IFN gene ex vivo. Each patient had lesions accessible
to intratumoral injection and distant lesions evaluable to measure systemic response.
Patients were entered into 1 of 3 treatment arms. The dose of the gene-transduced vaccine
was identical for each arm (1.5 X 108 PFU/dose administered at a volume of 0.3 mL for 5
consecutive days). Preclinical data suggested that gene transduction efficiency varied
depending on the cation (hexadimethrine bromide or protamine sulfate) mixed with the
vector during injection. In this trial, 4 patients received hexadimethrine bromide, 4 patients
received protamine sulfate, and 5 patients received no cation (36). Successful insertion of
the gamma-IFN gene into the tumor cell was based on the enzyme-linked immunospot and
polymerase chain reaction assays and was confirmed in each arm. Toxicity to the vector was
not observed, and there was no evidence of viable retrovirus contamination (65 samples
tested). Coadministration with cations did not appear to alter transgene expression following
injection into the tumors. Patients with evidence of transgene expression had a median
survival of 528 days compared with patients who had no evidence of transgene expression
(median survival, 333 days).
From this study we concluded that treatment via a single intratumoral injection was safe.
Efficacy evaluation was limited by the removal of the gene-transduced tumor 8 days after
injection in order to assess transgene expression. No responses in injected or distal lesions
were observed; however, prolonged survival of patients with identifiable transgene
expression was interesting. Further clinical research using multiple courses of therapy,
combination therapy with systemically administered cytokines (IL-2), and/or treatment of
patients with earlier stages of less extensive disease is indicated.
GAMMA-IFN RETROVIRAL VECTOR ADMINISTERED INTRATUMORALLY
WITH MULTIPLE COURSES IN PATIENTS WITH METASTATIC MELANOMA
Multiple courses of direct intratumoral injection of adenoviral and retroviral vector
preparations have been shown to be effective in the induction of regional tumor regression
in several mouse tumor models (37–44) and in patients receiving retroviral vectors
involving the p53 gene (45).
In our third trial with gamma-IFN retroviral vector in patients with metastatic melanoma,
we administered multiple intratumoral injections of gamma-IFN retroviral vector at
escalating doses without removing the injected lesion for a single 5-day injection course
(group A, n = 9) or for multiple 5-day injection courses (group B, n = 8). One cycle
consisted of doses of 0.3, 0.5, and 1.5 mL per injection of gamma-IFN retroviral vector (1 X
107 PFU/mL) for 5 consecutive days. Patients received either 1 cycle or 6 cycles
administered every 2 weeks.
Enzyme-linked immunosorbent assays were performed to assess induction of tumor-specific
antibodies against 4 melanoma tumor cell lines (DM 92, DM 93, DM 252, and DM 400), 2
nonmelanoma cell lines, and nonmalignant tissue (fibroblast lines). Studies to determine
evidence of replication-efficient retrovirus were also performed from patient samples prior
to administration. No significant toxicity was observed, and 10 of the 17 patients treated
showed evidence of elevated antitumor antibody response titers following injection. These
were generally specific for the melanoma cell lines and were highest in patients who
received the higher dose levels (46). Two patients achieved a complete histologic response,
and 2 patients achieved a partial response of the injected lesion (>=50% reduction of disease
but not complete). Three of the 8 patients who received multiple doses of gamma-IFN
retroviral vector achieved a response compared with only 1 of 9 patients who received a
single cycle. Furthermore, survival in patients entered into the multicycle treatment regimen
suggested improvement compared with survival of patients who received only a single cycle
( Figure 4 , P = 0.027 log rank). Consistent with prior data, gamma-IFN retroviral vector
intratumoral injection was well tolerated, and there was no evidence of replicationcompetent retrovirus in the product or patient samples. These continue to be analyzed in
surviving patients >4 years after initiation of investigation with gamma-IFN retroviral
vector.
This trial is the first to attempt multiple courses of direct intratumoral injections of gammaIFN retroviral vector. Animal studies suggest that the antitumor effect related to gammaIFN retroviral vector injection is correlated with the number of injections and the titer of the
retroviral vector preparation (44, 46). Further research involving multiple injections is
indicated.
Systemic effects have been infrequent in phase I studies with immune-modulating gene
vectors, although results of animal studies have demonstrated systemic antitumor effects
following injections with immune-modulating genes, such as gamma-IFN, HLA-B7, or IL2, in association with improved survival (12, 14, 21, 24, 26, 44, 48). Data suggest that
transfer of gamma-IFN gene into tumor cells may alter tumor antigen expression and induce
a localized immune response. However, clinical effects of a systemic response may be
difficult to evaluate in patients with bulky advanced disease who have an expected survival
of <6 months. It takes several months to build up the immune response against the tumor.
Before the immune system sufficiently responds to the vaccine, the malignant progress is
still occurring and may override the patient's immune response if the disease is too far
advanced when immunotherapy is initiated. Thus, given the potential systemic activity to
local injection, many trial designs consider initial treatment with chemotherapy or treatment
of patients with early stage disease since toxicity is minimal.
OTHER IMMUNE-MODULATING VACCINE APPROACHES PERFORMED IN
THE MARY CROWLEY MEDICAL RESEARCH FACILITY
Studies exploring vaccines transduced with the GM-CSF gene (GVAX) using autologous
tumor cells and allogeneic cell lines are being explored in patients with prostate cancer and
non–small cell lung cancer. Also, plasmid DNA for HLA-B7 (Allovectin-7) complexed
with a cationic lipid is being examined in melanoma patients. Phase I investigation reveals a
low toxicity profile.
The GVAX vaccine has been studied (phase I) in patients with melanoma, renal cell cancer,
and prostate cancer. GVAX vaccine is an allogeneic vaccine, which does not require
surgical harvest of autologous tumor tissue. Currently, we are performing trials in patients
with hormonal naive prostate cancer and hormonal refractory prostate cancer in which the
GM-CSF gene is placed into allogeneic prostate cell lines containing antigens most likely to
be expressed in autologous tumors. Patients receive multiple intradermal injections of the
prostate GVAX vaccine for as long as stable disease is maintained or induced response is
observed. Gene delivery uses an adenoviral vector for GM-CSF as opposed to a retroviral
vector, which was used for the gamma-IFN trials described.
Trials investigating the HLA-B7 gene use a plasmid DNA complex with a cationic lipid
mixture. Lipid-complexed DNA plasmids have a higher transduction-efficiency rate than
retroviral vectors, but intracellular release of the transgene product from the lipid complex
may have some limitations. Trials investigating intratumoral injection of Allovectin-7
include phase II studies in patients with refractory progressive melanoma and a phase III
investigation comparing patients receiving Allovectin-7 combined with chemotherapy
(dacarbazine) with patients receiving chemotherapy alone. Response rates of 35% in
injected lesions and 15% in systemic (not injected) lesions were observed in phase I/II trials.
CONCLUSION
Gene therapy offers a unique opportunity to modulate the immune system and potentially
enhance antitumor effects. Regardless of the delivery vehicle used, safety has been
confirmed with these approaches. Phase III trials are ongoing nationally and are being
performed at the Mary Crowley Medical Research Center to determine whether or not gene
therapy offers an advantage over standard treatment. Additionally, combining these
approaches with standard approaches may also benefit patients with advanced cancer.
Investigation of immune enhancement is one of several clinical research areas we are
pursuing.
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