 |
|
Combination Nonviral Interleukin 2 and Interleukin 12 Gene Therapy for Head and Neck Squamous Cell Carcinoma
Daqing Li, MD;
Elizabeth Shugert, MD;
Ming Guo, PhD;
Jeffrey S. Bishop, MS;
Bert W. O'Malley, Jr, MD
Arch Otolaryngol Head Neck Surg. 2001;127:1319-1324.
ABSTRACT
| |
Objective To determine the feasibility and efficacy of combination nonviral lipid-formulated
murine interleukin 2 (mIL-2) and polymer-formulated murine interleukin 12
(mIL-12) gene therapy for head and neck squamous cell carcinoma (HNSCC) in
a murine model.
Methods Randomized, controlled studies in a murine HNSCC model. Tumors were
established in the floor of mouth in C3H/HeJ immunocompetent mice. Established
tumors were directly injected with lipid-formulated mIL-2 and polymer-formulated
mIL-12 alone and in combination. Antitumor responses, cytokine expression,
and natural killer cell and cytolytic T-lymphocyte activity were assayed.
Results The use of combined mIL-2 and mIL-12 gene therapy resulted in significant
antitumor effects, compared with each of the single-cytokine and no-treatment
(control) groups (P = .01 to P = .02). Tumors treated with the formulated cytokine genes showed an
increased level of the corresponding proteins and decreased level of transforming
growth factor ß (TGF-ß) expression. Combined mIL-2 and mIL-12 treatment
consistently produced the greater activation of cytolytic T-lymphocyte and
natural killer cells than did single-cytokine treatment or other controls
at all concentrations tested. Augmented immune responses correlated with clinical
antitumor effects.
Conclusions The nonviral gene delivery system was well tolerated, and combined mIL-2
and mIL-12 gene transfer generated potent antitumor immune responses against
HNSCC in our murine model. Combined nonviral IL-2 and IL-12 gene therapy may
have great potential as a primary or adjuvant treatment for HNSCC in humans.
INTRODUCTION
HEAD AND NECK cancer afflicts 50 000 new patients each year in
the United States and more than 500 000 worldwide.1
During the past 30 years, the 3- to 5-year survival rate of patients with
advanced T3 and T4 squamous cell carcinoma of the head and neck (HNSCC) has
remained poor (20%-30%), despite considerable advances in surgical technique
and radiation delivery and improvement in chemotherapeutic strategies. This
reality has stimulated the development of novel therapeutic strategies for
primary or adjuvant treatment of this disease.2
Immunotherapy represents a particularly promising treatment strategy for patients
with HNSCC.
Interleukin 2 (IL-2) is naturally produced by T cells and serves as
an important growth and activation factor for cytolytic T lymphocytes (CTLs),
macrophages, natural killer (NK) cells, and B lymphocytes. Treatment with
IL-2 has produced definite tumor regression in patients with advanced cancer
such as renal cell carcinoma, melanoma, and colorectal cancer.3
In addition, evidence exists of local and systemic activation of immune cells
by peritumoral injections of IL-2 in patients with HNSCC.4-5
In addition to IL-2, interleukin 12 (IL-12) has been shown to have potent
antitumor efficacy.6-8
Produced by macrophages and dendritic cells, IL-12 enhances the cytolytic
function of NK cells and CTL, and it activates NK and T cells to secrete interferon 
(IFN-), a potent activator of macrophages. It has been demonstrated
that IL-12 gene therapy prevents the establishment of SCC, inhibits tumor
growth, and elicits long-term antitumor immunity.9
It has been well documented that the host immune system plays a major
role in recognition and destruction of tumor cells, and locoregional immunosuppression
allows the advancement of tumors.4, 10-12
This immunosuppression is characterized by depressed mitogen responsiveness
and re duced cytolytic activity, and by decreased cytokine production of tumor-infiltrating
lymphocytes relative to regional lymph node and peripheral blood lymphocytes.13-14 Transforming growth factor ß
(TGF-ß) produced by tumor cells is one of the most potent immunosuppressive
factors characterized to date. It has been shown to inhibit immunoregulatory
cytokine production and to suppress the proliferative response of T cells
to IL-2.15-16 Recently, Matthews
et al17 reported the restoration of immunogenicity
in prostate cancer cells by down-regulation of TGF-ß production.
The objective of this study was to demonstrate the efficiency of nonviral
gene transfer and to use this system to establish the superiority of combined
IL-2 and IL-12 gene therapy combined with single-cytokine therapy in the treatment
of HNSCC. Independently, IL-2 and IL-12 have been shown to have significant
antitumor effects, and evidence exists that IL-2 and IL-12 may be more effective
at inducing tumor rejection when given together.18-19
MATERIALS AND METHODS
PLASMIDS
The following 3 plasmids were used in this study: pIL0555, pIN0961,
and pVC1157. All of the plasmids contained the kanamycin resistance gene.
The expression cassette for murine IL-2 (mIL-2) was contained in pIL0555 with
the cytomegalovirus promoter. Two complete and separate transcription units,
1 for each of the subunits p35 and p40 that combined to form the biologically
active murine IL-12 (mIL-12) p70 molecule, were found in pIN0961. The transcription
unit for each subunit contained the cytomegalovirus promoter. We used pVC1157
as a control, containing no coding sequences. The plasmids were propagated
in Escherichia coli strain DH5 , purified using
alkaline lysis and column chromatography, and tested for endotoxin contamination
using an amebocyte assay (Limulus; BioWhittaker, Walkersville, Md).
FORMULATIONS
The mIL-2 plasmid (pIL0555) and control plasmid (pVC1157) were formulated
in the cationic lipid N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA; Avanti Polar Lipids,
Alabaster, Ala), with cholesterol as a colipid to optimize plasmid delivery.
Small unilamellar vesicles of a 1:1 molar ratio of DOTMA and cholesterol (Avanti
Polar Lipids) were prepared by means of microfluidization. Plasmid lipid complexes
were prepared by mixing purified plasmid with these liposomes under controlled
conditions in a solution containing 10% lactose as an isotonic agent. The
final plasmid-lipid mixture was formulated at a 0.25-mg/mL concentration of
plasmid DNA at a DNA-lipid charge ratio of 1:0.5 (-/+). The mIL-12 (pIN0961)
and control (pVC1157) plasmids were formulated in 5% polyvinylpyrrolidone.
The plasmid DNA was mixed in a 1:17 mass ratio with polyvinylpyrrolidone at
a final 1.92-mg/mL concentration of plasmid DNA.
ANIMAL MODEL
The animal model used for this study was a syngeneic orthotopic murine
model for HNSCC.20 The care and use of all
animals was in accordance with the guidelines of the animal welfare committee
of University of Maryland School of Medicine, Baltimore. To establish a tumor
model in the floor of the mouth, mice were injected with 1 x 105 SCC VII cells using a 25-gauge needle. Injections were made into the
floor of the mouth through the neck skin using a sterile technique at day
0. After day 5, established tumors were surgically exposed and measured in
3 dimensions using calipers, followed by intratumoral multidirectional injections
of the respective gene transfer per assigned treatment group. A second treatment
via percutaneous injection was given 4 days after first gene delivery. Animals
were killed 8 days after initial gene transfer treatment (on day 13 of the
experiment), and tumor size was again measured in 3 dimensions using calipers.
Harvested tumors, local lymph nodes, and spleens from each animal were used
for designated studies.
MEASUREMENT OF CYTOKINE EXPRESSION IN TUMOR EXPLANTS
The measurement of cytokine expression in tumor explants has been published
previously.21 In brief, the harvested and finely
minced tumors were cultured in 1 mL of Dulbecco Modified Eagle Medium (Sigma-Aldrich
Corp, St Louis, Mo) with 10% fetal bovine serum in 3.8-cm2 wells.
Medium was extracted after 24 hours, and cytokine assays were performed using
commercially available monoclonal antibody enzyme-linked immunosorbent assays
(ELISAs) for mIL-2, mIL-12 (p70), murine IFN- (R & D Systems, Minneapolis,
Minn), and TGF-ß1 (Promega, Madison, Wis).
NK-CELL ASSAY FROM SPLENOCYTES
Spleens were harvested and crushed to obtain splenocytes. Cells were
washed in Hank balanced salt solution, centrifuged, and resuspended in the
solution. Splenocytes were separated on Ficoll-Hypaque and again centrifuged
before resuspension in CTL medium. Yac-1 target cells were labeled with sodium
chromate Cr 51 (51Cr) and coincubated with 4 dilutions of effector
cells to yield 4 different effector-target cell ratios (100:1, 33:1, 10:1,
and 3:1). The resulting supernatant was extracted and measured on a -radiation
counter 12 hours after coincubating in 96-well plates.
CTL ASSAY FROM REGIONAL LYMPH NODE
Regional neck lymph nodes were microscopically dissected from the animals
and prepared as previously described.21 With
the use of CTL media, lymphocytes were washed twice, then plated into a 24-well
plate at a concentration of 4 x 106 cells/well. Mitomycin-treated
SCC VII cells were used as stimulator cells and plated into each of the wells
at a concentration of 1 x 105 cells/mL of media. Murine IL-2
(Pharmingen, San Diego, Calif) was then added to each well at a concentration
of 1 ng per well, and the cells were incubated for 7 days. The SCC VII target
cells were prepared by means of incubation for 1 hour with 51Cr,
followed by 3 washes in culture medium. The 51Cr-labeled SCC VII
cells were seeded into 96-well plates at a density of 3 x 103
cells per well containing 100 µL of medium. Effector lymphocytes then
were added to give effector-target ratios of 3:1, 10:1, 33:1, and 100:1, in
a final volume of 200 µL/well. Anti-CD4 or -CD8 blocking antibody (Pharmingen)
experiments were performed to assess the specificity of the tumor response.
Antibodies were added at a concentration of 0.2 µg/10 µL for each
tissue culture well, and blocking assays were performed in triplicate. Plates
were incubated for 16 hours and centrifuged, and the supernatant was assayed
for 51Cr release using a -radiation counter. The percentage
of specific lysis was determined using the following formula:
STATISTICAL ANALYSIS
The significance of differences between treatment groups was determined
by Mann-Whitney analysis.
RESULTS
DOSE RESPONSE OF FORMULATED mIL-2 AND mIL-12 GENE TRANSFER
We divided C3H/HeJ mice with established floor of the mouth tumors into
the following 3 experimental treatment groups: lipid-formulated mIL-2, polymer-formulated
mIL-12, and no treatment. The mIL-2 and mIL-12 treatment groups were further
divided into 3 subgroups and each animal received variable concentration of
treatment. Lipid-formulated mIL-2 was given at 3 different doses of 6.25,
12.5, and 25 µg, whereas polymer-formulated mIL-12 was used at 3 doses
of 48, 96, and 150 µg. All the animals received 2 injections, the first
during surgical exploration of the initial tumor and a subsequent injection
on day 4 after first gene delivery. All the animals were observed carefully
with attention to change in tumor size, and animals were humanely killed on
day 8 after the first injection. The tumor size in each animal was determined
using 3-dimensional caliper measurements. As shown in Figure 1, the mIL-2 and mIL-12 treatment groups were significantly
more effective in delaying tumor progression than the no-treatment control
group (P = .03 to P = .04).
The antitumor efficacy of mIL-2 and mIL-12 improved with increasing concentration
of injected dose. The optimal concentrations for mIL-2 and mIL-12 were determined
to be 12.5 µg and 96 µg, respectively (Figure 1), and any higher concentration provided minimal improvement
in efficacy with no statistical significance (P>.80).
For further experiments in assessing the efficacy of combined mIL-2 and mIL-12,
the concentration was used as the optimal dosing for mIL-2 and mIL-12.
|
|
|
|
Figure 1. Change in squamous cell carcinoma
VII tumor volume 8 days after lipid-formulated murine interleukin 2 (mIL-2)
or polymer-formulated murine interleukin 12 (mIL-12) treatment at different
doses from one of the experiments. Boxes represent the mean tumor volume in
the various treatment groups (each group had 8 mice), whereas the bars represent
the SE. A, Tumor volume change with 3 different doses ranging from 6.25 to
25 µg after formulated mIL-2 gene transfer. B, Tumor volume change with
3 different doses ranging from 48 to 150 µg after formulated mIL-12
gene transfer.
|
|
|
INHIBITION OF TUMOR GROWTH
The greatest antitumor effect was observed with combination therapy
consisting of mIL-2 and mIL-12 (Figure 2).
Mann-Whitney analysis revealed statistical significance between combined mIL-2
and mIL-12 therapy and mIL-2 or mIL-12 therapy alone (P = .01 to P = .02). Combined mIL-2 and mIL-12
therapy and each single-cytokine therapy showed statistically significant
antitumor response when compared with the lipid- and polymer-formulated plasmid
group and with the no-treatment control groups (P
= .01 with combined therapy; P = .009 with single-cytokine
therapy). There was no statistical significance between the formulated control
plasmid and no-treatment control groups (P>.50).
There was also no statistical significance between single-cytokine and combined
mIL-2 and mIL-12 therapies (P>.50).
|
|
|
|
Figure 2. Antitumor effects of combined,
single-, and control cytokine gene therapy treatments. Treatment injections
were made on days 5 and 9 and animals were killed on day 13. Combined mIL-2
and mIL-12 treatment was significantly more effective than single-cytokine
(P = .01 to P = .02) or control treatments (P = .01). Boxes represent the mean tumor volume in the various treatment
groups, whereas the bars represent the SE. Plasmid control indicates polymer-formulated
plasmid (pVC1157) control. Other abbreviations are given in the legend to
Figure 1.
|
|
|
CYTOKINE EXPRESSION
Increased levels of mIL-2 were found in the mIL-2 and combined mIL-2
and mIL-12 treatment groups, which showed statistical significance when compared
with mIL-12 alone and combined formulated plasmid and no-treatment control
groups (P<.05) (Figure 3A). Similarly, the greatest mIL-12 expression was seen in
the single mIL-12 and combined mIL-2 and mIL-12 groups (P<.05) (Figure 3B). Secondary
cytokine expression of murine IFN- was demonstrated in the combined
mIL-2 and mIL-12 treatment group and in the mIL-2 and mIL-12 treatment groups,
with statistical significance between these groups and the combined formulated
plasmid and no-treatment control groups (P<.05)
(Figure 3C). Decreased levels of
TGF-ß, a marker for tumor activity, were found in the combined mIL-2
and mIL-12 group and in the single-cytokine treatment groups, with significance
when compared with combined formulated plasmid and no-treatment control groups
(P<.05) (Figure
3D). There was no significant difference in decreased TGF-ß
expression between the combined- and single-cytokine therapies. Overall, assays
of cytokine expression demonstrated that formulated cytokine gene transfer
sufficiently induced the intended transgene expression and stimulated a desirable
secondary cytokine expression.
NK-CELL ACTIVITY
We harvested NK cells from splenocytes to evaluate their ability to
lyse tumor cells after the various treatment regimens. The greatest NK-cell
activity occurred in the combined mIL-2 and mIL-12 treatment group. At an
effector-target cell ratio of 100:1, combined mIL-2 and mIL-12 treatment resulted
in 38% target cell lysis. Single-cytokine therapy with mIL-2 or mIL-12 resulted
in 31% and 29% target cell lysis, respectively. The lipid- and polymer-formulated
and the no-treatment control groups resulted in limited NK-cell activity of
14% and 6%, respectively (Figure 4). An augmented antitumor NK-cell activity found in the combined mIL-2 and mIL-12
treatment group correlated well with the observation of inhibition of tumor
growth.
|
|
|
|
Figure 4. Representation of natural killer
(NK)–cell assays performed on splenocytes harvested 8 days after various
gene therapies. Combined mIL-2 and mIL-12 treatment showed a significant increase
in NK-cell activity compared with single-cytokine treatment groups and controls.
Abbreviations are given in the legends to Figure 1 and Figure 2.
|
|
|
CTL ACTIVITY
To evaluate regional cell-mediated immune responses in the animal model
with HNSCC, lymphocytes were obtained from the local lymph nodes and tested
for their ability to lyse tumor cells in vivo. As shown in Figure 5A, the greatest CTL activity occurred in the combined mIL-2
and mIL-12 treatment group. At an effector-target cell ratio of 100:1, the
combination group resulted in 40% target cell lysis. Single-cytokine therapy
with mIL-2 and mIL-12 resulted in 35% and 34% CTL activity, respectively.
Low levels of CTL activity were found in the lipid- and polymer-formulated
and the no-treatment control groups (15% and 10% cell lysis, respectively).
The results of CTL activity correlated well with the data for inhibition of
tumor growth and NK-cell activity.
|
|
|
|
Figure 5. Representation of cytolytic T-lymphocyte
(CTL) assays performed on lymphocytes harvested from lymph node dissections
8 days after gene therapy. A, Comparison of combined mIL-2 and mIL-12 gene
therapy with single-cytokine gene therapy and controls at 4 effector-target
cell dilutions. Combined mIL-2 and mIL-12 treatment showed a significant (P<.01) increase in CTL activity compared with single-cytokine treatment
groups and controls. B, Antibody blockade of CD4+ and CD8+ T cells demonstrating a primarily CD8+-mediated tumor cell
lysis. Abbreviations are given in the legends to Figure 1 and Figure 2.
|
|
|
To determine whether the antitumor immunity was associated with the
presence of tumor-specific CD8+ CTLs and/or CD4+ helper
T lymphocytes, monoclonal antibodies against CD8 or CD4 were incorporated
as blocking reagents in each CTL assay. As seen in Figure 5B, the monoclonal antibody against CD4 was ineffective in
blocking cell lysis. This result may indicate that the antitumor response
observed was primarily mediated by CD8+ CTLs.
COMMENT
Interleukin 2 and IL-12 are known to have antitumor properties, and
evidence exists that, given in combination, these cytokines will afford greater
effects.22 We observed the greatest antitumor
effect with combination mIL-2 and mIL-12 therapy, and these results support
the proposed additive strength of mIL-2 and mIL-12.
The superior antitumor activity of mIL-2 and mIL-12 when given together
may be explained by augmented NK-cell and CTL responses. The underlying mechanism
of the observed augmented NK-cell and CTL responses is thought to be related
to an increase in the expression levels of mIL-2, mIL-12, and secondary cytokine
IFN-, resulting in activation of local and systemic immune cells. It
has been known that IL-2 is a major T-cell growth and activation factor and
a potent growth and activation factor for NK cells. Numerous studies have
shown IL-2–activated tumor inhibition in vivo.21, 23
Interleukin 12 produced by macrophages and dendritic cells enhances the cytolytic
function of NK cells and CTLs. It has been shown that intratumoral delivery
of IL-12 results in increased infiltration of NK cells and CD4+
and CD8+ T cells and up-regulation of major histocompatibility
complex class I molecules in tumors and lymph nodes.24-25
Induction of secondary cytokines such as IFN- is also believed to augment
the antitumor immune response and incite a cascade in which activated lymphocytes
add to the stimulus by increasing their cytokine production. It would therefore
be advantageous if IL-2 and IL-12 could be used in combination.
In addition to increased cytokine expression, decreased TGF-ß expression
may contribute to the superior antitumor effects of combined IL-2 and IL-12
therapy. Transforming growth factor ß is a potent immunosuppressive cytokine.
Recent study has shown that down-regulation of TGF-ß production restores
immunogenicity in prostate cancer cells.17
Results of the present study have demonstrated the greatest reduction in TGF-ß
expression with mIL-2 and mIL-12 combination therapy, which correlates well
with tumor growth inhibition. The observed inhibition in tumor growth probably
results from overcoming the immunosuppression and reactivating suppressed
tumor-specific CTL in our mouse model with HNSCC.
With the role of cytokines in tumor regression established, an effective
protocol for local and sustained delivery to the tumor is needed for clinical
application. Systemic administration of lymphokines at pharmacological doses
produces high concentrations of lymphokines in the vasculature and suboptimal
levels at the local tumor site, resulting in limited antitumor effects but
moderate to severe toxic effects such as fever, chills, headaches, and capillary
leak syndrome.26 As a result of these limitations,
strategies have been explored to use viral vector systems for cytokine gene
delivery. Although adenoviral vectors have proven efficient in transferring
genes into target tissues and do not require active cell division for gene
uptake and expression, they induce antiviral immune responses and may generate
toxic effects from systemic dissemination.27
The use of retroviral vector systems is limited by the danger of cotransferring
contaminating infectious and potential transforming viruses. Given these limitations,
nonviral systems have been studied as an alternative method of gene delivery
in vivo.28
The major criticism and limiting factor of nonviral systems has been
the low efficiency of in vivo gene transfer compared with viral strategies.
One objective of this study was to demonstrate the efficiency of nonviral
gene transfer using a unique plasmid-based therapy specifically designed to
facilitate diffusion of the plasmid as well as gene uptake and expression.
Our results demonstrate significant cytokine expression and antitumor effect
after this nonviral therapy using lipid-formulated mIL-2 and polymer-formulated
mIL-12. This nonviral delivery system to the local tumor mass has proven to
be efficient in its gene transfer and, in addition, to circumvent the severe
toxic effects of systemic therapy and the limitations of viral-based gene
therapy.
CONCLUSIONS
The present study provides evidence that the nonviral gene delivery
system is well tolerated and further demonstrates that combined mIL-2 and
mIL-12 gene transfer generates potent antitumor immune responses against HNSCC
in our murine model. The results indicate that combined nonviral IL-2 and
IL-12 gene therapy may have great potential as a primary or adjuvant treatment
for HNSCC. The nonviral gene delivery system is a promising new tool that
warrants further laboratory investigation.
AUTHOR INFORMATION
Accepted for publication June 12, 2001.
Corresponding author and reprints: Bert W. O'Malley, Jr, MD, Division
of Otolaryngology–Head and Neck Surgery, 16 S Eutaw St, Suite 500, Baltimore,
MD 21201 (e-mail: bomalley{at}smail.umaryland.edu).
From the Department of Otolaryngology–Head and Neck Surgery (Drs
Li, Shugert, Guo, and O'Malley) and The Greenebaum Cancer Center (Drs Li and
O'Malley), University of Maryland School of Medicine, Baltimore; and Valentis,
Inc, The Woodlands, Tex (Mr Bishop). Dr O'Malley has a sponsored research
agreement with equity options in Valentis, Inc.
REFERENCES
| |
1. Landis SH, Murray T, Bolden S, et al. Cancer statistics. CA Cancer J Clin. 1998;48:6-29.
ABSTRACT
2. Suen JY, Myers EN. Head and Neck Cancer. New York, NY: Churchill Livingston Inc; 1981.
3. Rosenberg SA, Yang JC, White DE, et al. Durability of complete responses in patients with metastatic cancer
treated with high-dose interleukin 2: identification of the antigens mediating
response. Ann Surg. 1998;228:307-319.
FULL TEXT
|
ISI
| PUBMED
4. Whiteside TL, Letessier E, Hirabayashi H, et al. Evidence for local and systemic activation of immune cells by peritumoral
injections of interleukin 2 in patients with advanced squamous cell carcinoma
of the head and neck. Cancer Res. 1993;53:5654-5662.
FREE FULL TEXT
5. Cortesina G, De Stefani A, Majore L, et al. Loco-regional treatment with low and high doses of interleukin 2 of
head and neck squamous cell carcinoma recurrences [in Italian]. Acta Otorhinolaryngol Ital. 1994;14:3-9.
6. Nastala CL, Edington HD, McKinney TG, et al. Recombinant IL-12 administration induces tumor regression in association
with IFN- production. J Immunol. 1994;153:1697-1706.
ABSTRACT
7. Brunda MJ, Gately MK. Antitumor activity of interleukin 12. Clin Immunol Immunopathol. 1994;71:253-255.
FULL TEXT
|
ISI
| PUBMED
8. Taha MJ, Luistro L, Warrier RR, et al. Antitumor and antimetastatic activity of interleukin 12 against murine
tumors. J Exp Med. 1993;178:1223-1230.
FREE FULL TEXT
9. Myers JN, Mank-Seymour A, Zitvogel L, et al. Interleukin 12 gene therapy prevents establishment of SCC VII squamous
cell carcinomas, inhibits tumor growth, and elicits long-term antitumor immunity
in syngeneic C3H mice. Laryngoscope. 1998;108:261-268.
FULL TEXT
|
ISI
| PUBMED
10. Vitolo D, Letessier EM, Johnson JT, et al. Immunologic effector cells in head and neck cancer. J Natl Cancer Inst Monogr. 1992;13:203-208.
11. Cortesina G, Sacchi M, Galeazzi E, et al. Immunology of head and neck cancer: perspectives. Head Neck. 1993;15:74-77.
ISI
| PUBMED
12. Cortesina G, De Stefani A, Sacchi M, et al. Immunomodulation therapy for squamous cell carcinoma of the head and
neck. Head Neck. 1993;15:266-270.
ISI
| PUBMED
13. Mickel RA, Kessler DJ, Taylor JM, et al. Natural killer cell cytotoxicity in the peripheral blood, cervical
lymph nodes, and tumor of head and neck cancer patients. Cancer Res. 1988;48:5017-5022.
FREE FULL TEXT
14. Wang MB, Lichtenstein A, Mickel RA. Hierarchical immunosuppression of regional lymph nodes in patients
with head and neck squamous cell carcinoma. Otolaryngol Head Neck Surg. 1991;105:517-527.
ISI
| PUBMED
15. Espevik T, Figari IS, Shalaby MR, et al. Inhibition of cytokine production by cyclosporin A and transforming
growth factor ß. J Exp Med. 1987;166:571-576.
FREE FULL TEXT
16. Fontana A, Constam DB, Frei K, et al. Modulation of the immune response by transforming growth factor ß. Int Arch Allergy Immunol. 1992;99:1-7.
FULL TEXT
|
ISI
| PUBMED
17. Matthews E, Yang T, Janulis L, et al. Down-regulation of TGF-ß1 production restores immunogenicity in
prostate cancer cells. Br J Cancer. 2000;83:519-525.
FULL TEXT
|
ISI
| PUBMED
18. Pappo I, Tahara H, Robbins PD, et al. Administration of systemic or local interleukin 2 enhances the anti-tumor
effects of interleukin 12 gene therapy. J Surg Res. 1995;58:218-226.
FULL TEXT
|
ISI
| PUBMED
19. Wigginton JM, Komschlies KL, Back TC, et al. Administration of interleukin 12 with pulse interleukin 2 and the rapid
and complete eradication of murine renal carcinoma. J Natl Cancer Inst. 1996;88:38-43.
FREE FULL TEXT
20. O'Malley B, Cope KA, Johnson CS, et al. A new immunocompetent murine model for oral cancer. Arch Otolaryngol Head Neck Surg. 1997;123:20-24.
FULL TEXT
|
ISI
| PUBMED
21. Li D, Jiang W, Bishop J, et al. Combination surgery and nonviral interleukin 2 gene therapy for head
and neck cancer. Clin Cancer Res. 1999;5:1551-1556.
FREE FULL TEXT
22. Rashleigh SP, Kusher DI, Endicott JN, et al. Interleukins 2 and 12 activate natural killer cytolytic responses of
peripheral blood mononuclear cells from patients with head and neck squamous
cell carcinoma. Arch Otolaryngol Head Neck Surg. 1996;122:541-547.
ISI
| PUBMED
23. Whiteside TL, Chikamatsu K, Nagashima S, et al. Antitumor effects of cytolytic T lymphocytes (CTL) and natural killer
(NK) cells in head and neck cancer. Anticancer Res. 1996;16:2357-2364.
ISI
| PUBMED
24. Mendiratta SK, Quezada A, Matar M, et al. Intratumoral delivery of IL-12 gene by polyvinyl polymeric vector system
to murine renal and colon carcinoma results in potent antitumor immunity. Gene Ther. 1999;6:833-839.
FULL TEXT
|
ISI
| PUBMED
25. Kuriakose MA, Chen FA, Egilmez NK, et al. Interleukin 12 delivered by biodegradable microspheres promotes the
antitumor activity of human peripheral blood lymphocytes in a human head and
neck tumor xenograft/SCID mouse model. Head Neck. 2000;22:57-63.
FULL TEXT
|
ISI
| PUBMED
26. West WH, Tauer KW, Yannelli JR, et al. Constant-infusion recombinant interleukin 2 in adoptive immunotherapy
of advanced cancer. N Engl J Med. 1987;316:898-905.
ABSTRACT
27. Weichselbaum RR, Kufe D. Gene therapy of cancer. Lancet. 1997;349(suppl 2):SII10-SII12.
28. Parmiani G, Colombo MP, Melani C, et al. Cytokine gene transduction in the immunotherapy of cancer. Adv Pharmacol. 1997;40:259-307.
RELATED ARTICLE
Archives of Otolaryngology–Head & Neck Surgery Reader's Choice: Continuing Medical Education
Arch Otolaryngol Head Neck Surg. 2001;127(11):1403-1405.
FULL TEXT
THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES
Inhibition of NK Cell Activity through TGF-{beta}1 by Down-Regulation of NKG2D in a Murine Model of Head and Neck Cancer
Dasgupta et al.
J. Immunol. 2005;175:5541-5550.
ABSTRACT
| FULL TEXT
p12CDK2-AP1 Gene Therapy Strategy Inhibits Tumor Growth in an In vivo Mouse Model of Head and Neck Cancer
Figueiredo et al.
Clin. Cancer Res. 2005;11:3939-3948.
ABSTRACT
| FULL TEXT
Effects of Combined Therapy With Interleukin 2 and Interleukin 12 Gene-Transfected Tumor Vaccine for Head and Neck Carcinoma
Kimura et al.
Arch Otolaryngol Head Neck Surg 2003;129:1181-1185.
ABSTRACT
| FULL TEXT
Gene Therapy for the Treatment of Oral Squamous Cell Carcinoma
Xi and Grandis
J. Dent. Res. 2003;82:11-16.
ABSTRACT
| FULL TEXT
|
