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Trang 1Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
CANCER IMMUNOTHERAPY
Evade Immuno-Gene Therapy through Loss of
Sensitivity to IFN- γγγγγ Induced Apoptosis
Guillermo Mazzolini, Iñigo Narvaiza, Alfonso Martinez-Cruz,
Ainhoa Arina, Miguel Barajas, Juan Carlos Galofre, Cheng Qian,
Jose Maria Mato, Jesus Prieto, Ignacio Melero
1 Gene Therapy Unit FIMA, University of Navarra, Pamplona,
Navarra, Spain.
Combined injections into experimental tumor nodules of
adenovirus encoding IL-12 and certain chemokines are capable to
induce immune-mediated complete regressions In this study we
found that the combination of two adenoviruses, one encoding
IL-12 and other MIP3 α (AdCMVIL-12+AdCMVMIP3 α) was very
successful in treating CT-26 derived colon carcinomas However, in
experimental tumors generated from the pancreatic carcinoma cell
line Panc02 such combined treatment induces 50% of macroscopic
complete regressions, although local relapses within one week are
almost constant We derived cell lines from such relapsing tumors
and found that experimental malignancies derived from their inoculum
were not amenable to treatment in any case with
AdCMVIL-12+AdCMVMIP-3 α Importantly, relapsing cell lines were
insensitive to in vitro induction of apoptosis by IFNγ, in clear
contrast with the original Panc02 cells Comparative analyses by
cDNA arrays of relapsing cell lines versus wild type Panc02 were
performed revealing an important number of genes (383) whose
expression levels were modified more than two-fold These changes
grouped in certain gene ontology categories and should harbor the
mechanistic explanations of the acquired selective resistance to IFNγ
Interestingly, the expression at the RNA level of the
apoptosis-related protein clusterin is completely lost in the studied IFN
γ-resistant cell variants
Adenovirus To Induce Protein Production and
Reduce Tumor Burden
Mary Donahee,1 Karen Kozarsky,1 Deborah Welham.1
1 Protein Agents and Human Gene Therapy, GlaxoSmithKline,
King of Prussia, PA.
IL-1H4/IL-1F7 is a 22 kD cytokine in the Interleukin(IL)-1 family
that shares significant sequence homology with IL-18 Like IL-18,
IL-1H4 lacks a characteristic leader peptide but contains a propeptide
domain that is cleavable by members of the caspase family IL-18
has significant antitumor effects in mouse models; therefore,
IL-1H4 was evaluated as an antitumor agent Adenovirus vectors
encoding IL-1H4 and IL-18 were used to obtain sustained protein
expression in mice injected with a mouse sarcoma line Prior to in
vivo testing, verification of protein expression was performed in
vitro Lysates from A549, Huh-7 and HeLa cells that were transduced
with Ad.IL-1H4 showed both the 22 kD mature form of the IL-1H4
protein as well as the 26 kD pro form while only low levels of
secreted IL-1H4 were detected in the media In vivo, MCA205
tumor cells were introduced into C57Bl/6 male mice 2 days prior to
treatment with 1 x 10 11 viral particles of adenovirus The treatment
groups receiving the Ad.IL-1H4 or Ad.IL-18 showed a 5 day delay
in tumor growth over the control groups Subsequent experimentation
was performed using a dual infection with both Ad.IL-1H4 and
Ad.IL-18 Again a trend toward tumor growth reduction was seen
The group receiving Ad.IL-1H4 showed a 5 day delay in tumor
growth vs the control group while the Ad.IL-18 showed a 7 day
delay The group receiving both Ad.IL-1H4 and Ad.IL-18 also
showed a 7 day delay in tumor growth, indicating no obvious
synergistic effect when combining the viruses
Injection of TG1024 (Adenovirus-Interleukin-2) in Melanoma and Accessible Solid Tumours
Rochlitz Christoph,1 Morcinek Jessica,3 Reuter Juergen,1 Slos Philippe,2 Squiban Patrick,2 Dummer Reinhard.3
1 Medical Oncology, Kantonsspital, Basel, Switzerland;
2 Transgene, Strasbourg, France; 3 Dermatology, University Hospital, Zurich, Switzerland.
Many solid tumors cannot be completely removed by surgery and do not respond to radio or chemotherapy However, they are accessible to direct injection of agents such as gene therapy vectors
A percentage of some advanced solid tumors, melanoma and renal cell cancer respond to systemic Interleukin-2 (IL-2) and/or interferon However, these are associated with significant toxicities Local production of IL-2, by direct injection of a gene transfer agent, should result in local production of IL2, and associated local biological impact, but with lower systemic toxicities The TG1024 product is made of a suspension of non-replicating (E1 and E3 regions deleted) recombinant adenoviral particles containing a human IL-2 cDNA insert We undertook a phase I, open-label, dose-escalating trial of repeated intratumoral administration of TG1024
in patients with advanced melanoma and other accessible solid tumors Twenty patients (12 melanoma and 9 other solid tumors were enrolled in 5 successive cohorts at the following TG1024 doses: 3x10E8 total particles (tp), 3x10E9 tp, 3x10E10 tp, 8x10E10 tp and 3x10E11 tp Patients received intratumoral injections of TG1024 into the designated lesions on days 1, 21, and 42 (1 treatment cycle) and thereafter up to 4 cycles, if there was no evidence of progressive disease (PD) Blood samples were taken at baseline, before injection,
6 hours after injection and 1 week after injection and cytokine levels evaluated A dose dependent production of circulating IL2 was noted IL2 production was particularly striking in melanoma patients Repeated administration was associated with repeated cycles of detectable IL2 in the circulation of several patients Treatment was well tolerated Most common adverse event were injections site reactions Only 2 grade III (fever, transient lymphopenia) adverse events were noted and 3x10E11 tp has been considered as the maximum tolerated dose Evaluations of the patients clinical responses are still are ongoing and will be presented Our results show that local administration of an IL2 gene transfer agent can result in the production of significant levels of IL2, but does not result in the severe toxicities associated with systemic administration
of IL2 This information is of importance in the design of new gene delivery-based therapeutics in cancer
Principal Investigator and Consultant
Adenovirus-Interferon- γγγγγ (TG1042) in Primary Cutaneous T and B Cell Lymphomas
Urosevic Mirjana,1 Mayer Tania,1 Morcinek Jessica,1 Acres Bruce,2 Slos Philippe,2 Squiban Patrick,2 Burg Gunter,1 Dummer Reinhard.1
1 Dermatology, University Hospital, Zurich, Switzerland;
2 Transgene, Strasbourg, France.
During the progression of primary cutaneous lymphomas (CL), production of the Th1 cytokines decreases with a shift towards the immunosuppressive Th2 phenotype This group of diseases has been therefore successfully treated with interferons (IFNs), counterbalancing the Th2-skewing state We undertook a phase I, open-label, dose-escalating trial of repeated, intratumoral administration of TG1042 in patients with advanced primary cutaneous T cell lymphomas (CTCL) and multilesional cutaneous
B cell lymphomas (CBCL) TG1042 product is a suspension of non-replicating (E1 and E3 regions deleted) recombinant adenoviral
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Copyright © The American Society of Gene Therapy
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CANCER IMMUNOTHERAPY
particles containing human IFN-γ cDNA insert Nine patients (7
CTCL, 2 CBCL) were enrolled in 3 successive cohorts at the
following TG1042 doses: 3x10E9 total particles (tp), 3x10E10 tp
and 3x10E11 tp Patients received intratumoral injections of TG1042
into the designated lesions on days 1, 8, and 15 with no injection in
the fourth week (1 treatment cycle) and thereafter up to 4 cycles, if
there was no evidence of progressive disease (PD) The injected
lesion was biopsied at baseline and after 3 injections and assessed
for changes in lesion’s morphology, immunohistochemical changes
of T and B-cell (CD3, 4, 8, 20, 21, 79a), NK (CD56), cytotoxic
(TIA-1), and dendritic cell markers (CD1a) as well as for the
expression of coxsackie-adenovirus receptor (CAR) and HLA class
I molecules In addition, the lesions were evaluated for the expression
of transgene-derived IFN-γ as well as for total cytokine production
(IFN-γ, IL-2, IL-13, IL-10) with respect to CD4 and CD8 T cells
population by quantitative PCR Clinical responses and stabilizations
were observed in the majority of the patients Injection site reaction
was the most commonly observed adverse event Following TG1042
injections, histology demonstrated pronounced changes in infiltrate
pattern differing from initial lymphoma finding, with signs of
vasculitis and increase in eosinophil and neutrophil numbers
Immunohistochemistry revealed increase in CD8 and TIA-1
immunoreactivity in various cell types All lesions demonstrated
clear up-regulation of CAR following injection of TG1042
Quantitative PCR showed decrease in CD4/CD8 ratio in 6/8
patients Transgene-derived IFN-γ mRNA could be detected in
injected lesions, implying successful IFN-γ gene transfer Our results
reveal for the first time the in situ immunological changes following
the administration of adenoviral vector expressing IFN-γ in primary
CTCL and CBCL, offering new insight and information of importance
to the design of new gene delivery-based therapeutics in cancer
Principal investigator and consultant
Mediated by a Novel Cationic Liposome
Preparation in an Orthotopic Bladder Cancer
Model
Qinghui Wu,1 Ratha Mahendran,1 Kesavan Esuvaranathan.1
1 Surgery, National University of Singapore, Singapore.
Purpose: To assess cytokine gene expression by tumor cells in
vitro and in vivo in an orthotopic mouse bladder cancer model after
liposome-mediated gene transfer To evaluate the treatment effect of
cytokines on the growth of orthotopic tumor
Materials and Methods: The murine bladder cancer cell lines
MB49 was maintained in RPMI 1640 1 X 105 cells was instilled
into the bladder of C57BL/6 mice after electric cautery to establish
the tumor model The plasmid vectors were constructed by inserting
the coding sequences of IFN-α1 and GM-CSF into plasmid vector
pBudCE4.1 Transient transfection was performed using a cationic
lipid DOTAP and methyl-b-cyclodextrin solubilized cholesterol
(MBC) The expression level of IFN-α1 and GM-CSF in culture
medium was checked by ELISA The effects of cytokine gene transfer
on tumor cell proliferation and some surface marker expression such
as MHC-I and II were evaluated The presence of the tumors was
confirmed by histological examination and MRI scan of the mouse
bladders The expression of the transgene in situ was confirmed by
immunohistochemistry and x-gal staining Four groups of animals
with orthotopic bladder tumor were treated with control vector,
GM-CSF, interferon-alpha or both cytokines respectively twice a
week by intravesical instillation of the gene tranfer mixture
Results: Superficial bladder tumors were consistently established
by intravesical instillation of MB49 cells The tumors were detectable
by MRI scan The expression levels of both cytokines in transfected
cell lines were increased significantly The inhibition of in vitro
tumor cells proliferation and up-regulation of some surface markers
were observed In situ gene transfer to bladder tumors was accomplished via intravesical instillation of plasmid DNA/DOTAP/ MBC beta-galactosidase (b-gal) after a single 2h in situ transfection Using immunohistochemistry, it could be clearly seen that cytokine was produced by the urothelial cells The survival of animals in treatment group was increased as compare to control group, some
tumors were even cured Conclusion: The orthotopic bladder cancer
model is very useful tool to study the feasibility of potential cytokine gene therapy We demonstrated here that the orthotopic mouse bladder cancer can be transfected with a single instillation of liposome-DNA complexes The results also suggest that our liposome-mediated cytokine transfection system appears to be a potential promising non-viral gene treatment modality to bladder
cancer in vivo.
Expression in Mammary Tumors Modulates Multiple Malignant Phenotypes and Reduces Metastatic Burden
Van Tsai,1 Anja Muller,2 Albert Zlotnick,2 Brian Helmich,1 Iqbal Ahmed,1 Robert Ralston,1 Daniel Maneval,1 Drake LaFace.1
1 Canji, Inc., a Division of Schering-Plough, San Diego, CA;
2 DNAX Research Institute, Palo Alto, CA.
We report here the use of recombinant adenoviral (rAd) vectors
to induce expression of human IL-10 in established tumors to examine the capacity for suppressing tumor growth and spontaneous metastasis in a murine mammary carcinoma model Prior reports of the effect of IL-10 expression on the metastatic phenotype have relied on transfection or pre-transduction prior to tumor implantation Thus IL-10 expression had preceded the tissue responses required for establishing a solid tumor and promoting metastasis Treatment
of established tumors with rAd-empty control vector resulted in modest tumor growth inhibition, whereas, tumor regression was observed in the rAd/IL-10 treated mice In Addition, spontaneous metastasis to axillary lymph nodes and lungs was markedly inhibited
in rAd/IL-10 treated mice Immunohistochemical analysis revealed markedly reduced CD31 expression in rAd/IL-10 treated tumors but not in rAd/empty vector control treated tumors Moreover,
rIL-10 inhibited endothelial cell migration induced by activated macrophages These results support the hypothesis that IL-10 can inhibit tumor metastasis by suppressing macrophage-mediated induction of angiogenesis Additional mechanisms for inhibition of tumor growth and metastasis associated with expression of IL-10 were elucidated using a TaqMan RT-PCR based molecular profiling method We performed an analysis of differential expression profiles
of 121 genes for interdependent expression profiles associated with IL-10 expression that correlated with the capacity to suppress metastasis Treatment with rAd/empty control vector induced very few changes in gene expression, whereas, rAd/IL-10 modulated expression of numerous genes that regulate tissue remodeling, angiogenesis and immune responses Importantly, IL-10 suppressed gene expression of several key metalloproteinases and pro-metastatic cytokines that have been demonstrated to promote tumor progression and metastasis in breast carcinomas The capacity of rAd/IL-10 to modulate expression of multiple mediators of this complex multi-step progression may circumvent the conundrum of redundant molecular mechanisms promoting malignant phenotypes