59 Interferon Lambda Suppresses the Growth of Human Esophageal Carcinoma Cells and Enhances the Sensitivity to Anti Cancer Agents Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The Am[.]
Trang 1Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
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fl ank followed by twice-weekly injection of PEI nanoparticles into
the tail vein once palpable tumors were observed Control mice had
an average tumor volume of 284 mm3 at the time of sacrifi ce while
mice treated with 1.5 mg/kg of PEI:peIF5AK50R:h5A1 had an average
tumor volume of only 13 mm3, a 95 % (*p = 0.026) reduction in
tumor growth Furthermore, when we attempted to excise the tumors
after sacrifi ce, no residual tumors could be found under the skin of
the treated mice Established tumors (>100 mm3) also responded
to treatment with 1.5 mg/kg of PEI:peIF5AK50R:h5A1, resulting
in the average tumor volume falling from 133 mm3 to 30.5 mm3
after 24 days of treatment TUNEL-labeling revealed evidence of
apoptotic cells in the treated tumors indicating that tumor regression
occurs through the induction of apoptosis The biodistribution of
systemically-delivered PEI nanoparticles was determined using a
GFP plasmid and a fl uorescently-labeled (DY547) siRNA GFP
expression and DY547-siRNA uptake was observed in subcutaneous
tumor tissue as well as in cells of the bone marrow The safety of
long-term administration of PEI:peIF5AK50R:h5A1was assessed in
BALB/c mice Mice receiving twice-weekly administration of PEI
nanoparticles continued to gain weight over the nine-week study
period and no hematological or liver function changes were observed
In summary, our preclinical data indicate that it is effi cacious and
safe to use systemic administration of PEI:peIF5AK50R:h5A1nanopa
rticles in the treatment of multiple myeloma PEI nanoparticles are
taken up by cells of the bone marrow following systemic delivery
indicating that they may be feasible as a delivery vehicle for gene
therapy treatment of multiple myeloma patients
Vectors Expressing Syngeneic IFNα for Pancreatic
Cancer
Julia Davydova,1 Eric Brown,1 Selwyn Vickers,1 Masato
Yamamoto.1
1 Surgery, University of Minnesota, Minneapolis, MN.
Interferon α (IFN) which is commonly used as an antiviral
agent exhibits several properties that might be a great use for
combination therapy of cancer (e.g direct inhibition of tumor cell
growth and indirect antitumor effects through immunomodulation,
antiangiogenesis, radio- and chemo-sensitization) Despite
encouraging results for survival rates in clinical studies employing
IFN chemoradiation therapy, utilization of IFN-based regimen has
been impeded by systemic IFN toxicity, unreliable tumor delivery,
and its short half-life in blood circulation To reduce IFN toxicity
and enhance its effi cacy, gene therapy approaches, such oncolytic
adenoviral vectors (Ads) expressing human IFN can be used In this
work, we apply infectivity-enhanced conditionally replicative Ads
(CRAds) expressing syngeneic IFNα in replication-dependent manner
to treat pancreatic cancer To increase Ad potency, we enhanced the
effi ciency of cell lysis through overexpression of adenoviral death
protein and equipped the vector with 5/3 and RGD-modifi ed fi bers
To study antitumor and immunomodulatory effect of
replication-dependent IFN expression in syngeneic hamster and immunodefi cient
mouse models, we designed Ads expressing hamster and human IFNs
Comparison of vector functionality in vitro in hamster and human
pancreatic cancer cell lines revealed that oncolytic Ads expressing
IFN require approximately 3 orders lower titers to successfully kill
the cells compare to non-replicative IFN vectors Killing effect and
decline in cell viability observed with oncolytic IFN vector were
signifi cantly superior compared to that with identical counterpart
vectors without IFN The cytotoxicity effect correlated well with IFN
production in culture supernatant The IFN concentration increased
in a time- and dose-dependent manner after infection with oncolytic
Ad, indicating that IFN production depends upon viral replication
For instance, infection of MiaPaca2 cells with oncolytic Ad5/3IFN
at 0.1 vp/cell resulted in 2 order higher IFN level by day 12 post
infection compared to that of non-replicative IFN vector To study direct and indirect antitumor effect of IFN vectors, we established subcutaneous hamster HP1 tumors in immunocompetent Syrian hamsters and human MiaPaca2 xenografts in nude mice In hamsters, the tumors were treated with a single intratumoral injection of RGD-modifi ed vectors expressing hamster IFN Signifi cant antitumor effect was observed with both non-replicative and oncolytic IFN vectors Treatment with non-replicative vector was as effective as that with oncolytic Ad during the fi rst days after injection; however, after day
12 we observed tumor re-growth in the non-replicative vector group and by day 20 oncolytic Ad IFN signifi cantly outperformed the non-replicative vector Importantly, the hamsters treated with oncolytic IFN vector exhibited signifi cant tumor shrinkage compared to initial tumor size The antitumor effect in MiaPaca2 xenograft in nude mice was less evident compared to those in the HP1 model, suggesting possible indirect antitumor effect of IFNα as an immunomodulator These data indicate that infectivity-enhanced CRAd expressing syngeneic IFNα is a powerful therapeutic modality for pancreatic cancer
of Human Esophageal Carcinoma Cells and Enhances the Sensitivity to Anti-Cancer Agents
Quanhai Li,1 Kiyoko Kawamura,1 Guangyu Ma,1,2 Nobuo Suzuki,2 Yuki Takei,1,3 Naoto Yamaguchi,3 Fumi Iwata,4 Muneo Numasaki,4 Hideaki Shimada,5 Masatoshi Tagawa.1
1 Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan; 2 Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, Chiba, Japan; 3 Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan; 4 Department of Nutritional Physiology, Faculty of Pharmaceutical Science, Josai University, Sakado, Japan;
5 Division of Gastroenterological Surgery, Chiba Cancer Center, Chiba, Japan.
Novel cytokines, interleukin-28A (IL-28A), IL-28B and IL-29, have been identifi ed to belong to a novel interferon (IFN) family, IFN-lambdas All the three cytokines have similar functions and IFN-lambdas are classifi ed as a type III IFN IFN-lambdas could have a distinct activity compared with type I IFN, IFN-alpha and IFN-beta although the precise biological properties of IFN-lambdas remain to be uncharacterized We found that human esophageal carcinoma cell lines expressed the receptor complex of IFN-lambdas consisting of IL-10Rbeta and a novel molecule, IL-28R Recombinant IFN-lambda1 up-regulated the expression of level
of the class I molecules of the major histocompatibility complexes
(MHC) and induced the gene expression of myxovirus resistance A and 2’,5’-oligoadenylate synthetase, which are involved in antiviral
protection, in all the esophageal carcinoma cells tested We also found that IFN-lambda1 inhibited the proliferation of esophageal carcinoma cells due to multiple mechanisms including cell cycle arrest or apoptosis The cell cycle arrest was accompanied by up-regulated p21 gene expression and increased G0/G1 population Apoptosis was evidenced by increased subG1 population and Annexin-V-positive cells Although all the esophageal carcinoma cells expressed the IFN-lambdas receptor complexes and up-regulated MHC class I expression with IFN-lambda1, inhibited proliferation was not observed in all the cells tested Several human normal cells, positive for IFN-alpha receptor complexes, did not express one
of the IFN-lambda receptor complexes, and subsequently did not respond to IFN-lambda1 but were susceptible to IFN-alpha-mediated growth inhibition We demonstrated that combinatory use of 5-FU
or cisplatin with IFN-lambdas further enhanced the cytotoxicity
to sensitive esophageal carcinoma cells but not to normal cells Adenoviruses with type35 fi ber bearing the IL-28A gene induced
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CANCER-APOPTOSIS & SUICIDE/CANCER-IMMUNOTHERAPY,
ADOPTIVE T CELL THERAPY
suppressed cell growth of IFN-lambdas-sensitive carcinoma cells
These data collectively suggest that IFN-lambdas is a potential
anticancer agent for esophageal carcinoma and useful in combination
with chemotherapeutic agents
Receptor Targeting CD19 (CAR19), Optimized
Human IL-15, and iCaspase9 To Enhance the
Activity and Safety of Cytotoxic T Lymphocytes
Valentina Hoyos,1 Barbara Savoldo,1 Juan F Vera,1 Concetta
Quintarelli,1 Helen E Heslop,1 Cliona M Rooney,1 Malcolm K
Brenner,1 Gianpietro Dotti.1
1 Pediatrics, Medicine, Center for Cell and Gene Therapy, Baylor
College of Medicine, Houston, TX.
Recurrence of disease remains the largest cause of mortality
for many patients transplanted for relapsed or refractory B cell
malignancies Modifi cation of primary T cells to express a chimeric
antigen receptor (CAR) targeting CD19 represent an attractive
strategy to maintain remission after conventional treatment
However, their effi cacy is limited by poor expansion within the tumor
microenvironment The addition of co-stimulatory endodomains,
such as CD28, to the CAR may enhance cell expansion in response
to the antigen, but cell growth and survival remain suboptimal To
further potentiate the expansion and survival of CAR-modifi ed T
lymphocytes, we generated a new vector encoding 3 molecules:
CAR.19 incorporating the CD28 endodomain, codon optimized
hIL15 to enhance cell survival and growth, and an inducible suicide
gene based on the expression of Caspase9 (iCasp9) to increase the
margin of safety associated with transgenic expression of an autocrine
growth factor These three sequences were linked using 2A-like
peptide sequences We compared the proliferative capacity, cytotoxic
activity and in vivo anti-tumor effects of T lymphocytes expressing
either CAR.19-28ζ alone or CAR.19-28ζ, IL15 and the suicide gene
T lymphocytes were activated with OKT3/CD28 antibodies and then
transduced with retroviral supernatants Phenotypic analysis showed
70±10% and 75±5% transduction effi ciency for iCasp9/CAR19-28ζ/
IL15 and CAR19-28ζ T cells, respectively Only the
iCasp9/CAR19-28ζ /IL15 T cells produced IL15 (>100pg/mL) after stimulation with
CD19+ tumor cells T cells kept in culture for 4 weeks stimulated
weekly with CD19+ B-CLL cells were co-cultured with CD19+
Daudi cells After 72 hours iCasp9/CAR19-28ζ/IL15 T cells were
more effi cient eliminating tumor cells compared to CAR19-28ζ T
cells (0.7% residual tumor cells vs 14% respectively) Furthermore,
labeling with carboxyfl uorescein diacetate succinimidyl ester (CFSE)
showed that the proliferation of the iCasp9/CAR19-28ζ/IL15+ T cells
in response to CD19+ tumor cells was greater than that of CAR.19-28ζ
control+ T cells Finally, the activation of the suicide gene iCasp9
with a small-molecule dimerizer (CID 50ng/mL) rapidly induced
>90% apoptosis of T cells expressing iCasp9/CAR19-28ζ/IL15
Hence all three transgenes were functional To assess the antitumor
effects of the modifi ed cells in vivo, we used a xenograft SCID mouse
model and an in vivo bioluminescence system CD19+ Daudi cells
(1x106) expressing fi refl y Luciferase (FL) were injected i.p., and
on day 4, mice received i.p iCasp9/CAR19-28ζ/IL15+ or
CAR19-28ζ+ or control T cells (10x106) By day 30 the tumor signal was
signifi cantly reduced in mice receiving iCasp9/CAR19-28ζ/IL15+
T cells (ROI<7x107) compared to mice receiving CAR19-28ζ or
control T cells (ROI>2.0x109) In conclusion, our data indicate that
a tricistronic vector can effectively be expressed in tumor-redirected
human T cells, improving their survival and allowing their destruction
should unwanted effects occur
(CAR) Expression on Regulatory T Cells
Ibrahim Akalin,1 Serena K Perna,1 Biagio De Angelis,1 Fatma V Okur,1 Cliona M Rooney,1 Helen Heslop,1 Malcolm K Brenner,1 Barbara Savoldo,1 Gianpietro Dotti.1
1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX.
Adoptive immunotherapies with genetically modified T lymphocytes are endowed with a means for effectively treating malignant and infectious disorders Transduction of T cells with chimeric antigen receptors (CARs) can redirect the cellular immune response to almost any surface target antigen The function of CAR+
T cells in vivo may, however, be impaired by naturally occurring
regulatory T cells (Tregs) Moreover, Tregs themselves might be the inadvertent targets of CAR transfer, resulting in an undesired increase
in their presence at the site of activity of CAR-expressing effector T cells To discover the importance of this effect and to devise means
to avoid this, we isolated CD4+CD25bright Treg cells from peripheral blood of healthy donors and assessed their inhibitory function using carboxyfl uorescein diacetate succinimidyl ester (CFSE) dilution assay We then transduced peripheral blood polymorphonuclear cells (PBMCs) and Tregs with a chimeric antigen receptor targeting CD19 (CAR-CD19-CD28ζ), since this antigen is present on a high proportion of human B cell malignancies, and is the target for several current trials of T cell immunotherapy using such CAR-CD19 Our results showed that both activated PBMCs and Tregs are transduced and express CAR-CD19 (from 20% to 80%) While CAR-CD19+ T cells showed cytotoxic activity against CD19+ target cells as assessed
by 51Cr release assay (71%±34% at E:T ratio 20:1), CAR-CD19+ Tregs lacked effector function (6%±0.1% at E:T ratio 20:1) (p=0.01), but retained inhibitory activity in an MLR [% of proliferation were 68%±18% and 28%±19% when activated T cells were incubated with CD4+CD25- control cells and CAR-CD19+ Tregs, respectively (p=0.001)] More importantly, when CAR-CD19+ Tregs were mixed with CAR-CD19+ T cells (ratio 5:1) they inhibited the anti-tumor effects of these cells (% of remaining CD19+ tumor cells were 49%±21% and 2%±4% when tumor cells were co-cultured with CAR-CD19+ T cells with or without the addition of CAR-CD19+ Tregs) (p=0.02) To discover whether the inhibitory effect on CAR-CD19+ T cells by CAR-CAR-CD19+ Tregs was due to competition for the same epitopes on the CD19 target antigen, we redirected Tregs and
T cells with two different CARs (CAR-CD19 and CAR-CD30) We found that inhibition mediated by Tregs was maintained, suggesting that specifi c regulation rather than epitope/antigen competition was responsible for the observed inhibition Hence, transduction of unseparated peripheral blood T cells transduces both effector T cells and Tregs, and the latter are detrimental to the function of the former Future efforts to generate CAR-modifi ed T cells for human use may need to separate effector T cells from Tregs before transduction or before infusion
Specifi cally Target c-Met under Hypoxia
Sonny O Ang,1 Simon Olivares,1 Drew C Deniger,1 Dean A Lee,1 Richard E Champlin,1 Laurence J Cooper.1
1 University of Texas MD Anderson Cancer Center, Houston, TX.
Malignancies localized within chronic and intermittent hypoxic niches, is a negative prognosticator for many aggressive, chemo- and radio-resistant solid tumors The decreased oxygen tension, depleted nutrient levels, and low extracellular pH in this tumor microenvironment can limit T-cell viability, cytotoxicity, and thus immunotherapeutic effi cacy This makes T-cell mediated elimination
of bulky solid tumors diffi cult However, this also represents a micro-environment that can be exploited by genetic engineering to activate
T cells for redirected killing We developed a new approach that