509 Dose Escalation of Therapeutic High Capacity, Gutless Adenoviral Vectors in the Naïve Rat Brain Pre Clinical Safety and Toxicity Evaluation as a Prelude to a Phase I Clinical Trial for Glioma Mol[.]
Trang 1Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
S196
CANCER - IMMUNOTHERAPY II
Apoptosis was induced by upregulation of caspases 3, 8 and 9
Secondly, we compared the therapeutic effi cacy of anthocyanins
with a leukemia specifi c DNA vaccine to treat mice with minimal
residual disease (MRD) of syngeneic Ph+ ALL Non-viral minimalistic
immunogenically defi ned gene expression vectors (MIDGE) encoding
a BCR-ABL fusion specifi c peptide, GM-CSF and IL12 were used for
in vivo transfection of murine skin As TLR-9 agonist we used a
DNA-based double stem-loop immunomodulator (dSLIM), containing six
CpG-motifs Following a lethal leukemia challenge mice received
either daily oral applications of berry extract or intraperitoneal
applications of cyanidin from day 1 – 21 or were immunized with
the DNA vaccine on days 2 and 9 In addition, two groups received
a combination of the DNA vaccine and berry extract or cyanidin In
contrast to the anti-leukemia activity in vitro the anthocyanins were
not effi cient to treat mice with Ph+ ALL in vivo However, mice which
received the DNA vaccine showed a signifi cant longer tumor-free and
overall survival compared to the control and a survival rate of 56%
Intriguingly, the combined treatment with DNA vaccine and berry
extract but not with cyanidin further improved the outcome and lead
to a signifi cant longer tumor-free and overall survival compared to
the DNA vaccine alone and a survival rate of 90% In conclusion,
we provide data that a leukemia-specifi c DNA vaccine and berry
extract act synergistically in the treatment of mice with MRD This
approach may have implications to optimize maintenance therapy in
patients with Ph+ ALL
Cells during Activation
Vladimir Senyukov,1 Cecele J Denman,1 Laurence J N Cooper,1
Dean A Lee.1
1 Pediatrics, UT MD Anderson Cancer Center, Houston, TX.
Natural killer (NK) cells, as a key component of innate immunty,
have recently shown clinical potential for adoptive immunotherapy
against broad spectrum of oncological diseases One of the major
obstacles for adoptive NK cell immunotherapy is obtaining suffi cient
numbers of NK cells for effective therapy To address this hurdle, we
developed an artifi cial antigen presenting cells (aAPC) to expand
primary NK cells in vitro using K562 cells gene-modifi ed to express
membrane-bound cytokine, and showed that mIL21 promoted stronger
NK cells expansion than mIL15 To investigate this phenomenon we
compared NK cell proliferation kinetics (by SFCE dilution) in parallel
with daily assessment of apoptosis (by annexin V biding) in response
to mIL15 and mIL21 We fi nd that in the presence of mIL21 NK
cells proliferated much faster than in response to mIL15 (fi g.1a)
Moreover, in the presence of mIL15 as much as 90% of NK cells
(versus 15% in presence of mIL21) became able to bind annexin V
by the 3rd day after aAPC stimulation (fi g1b) Furthermore, we have
found that mIL21 up-regulated and supported CD160 expression by
NK cells during expansion time (42% in presence of mIL21 vs 6%
in response to mIL15 on day 3 and 83% vs 16% on day 7) CD160
is a GPI-anchored protein and its expression is down-modulated by
GPI-specifi c phospholipase D and synthesis of which, in turn, is
induced in NK cells in response to IL15 and IL2 It has been shown
that CD160 signaling mediates PI3K-dependent survival and growth
in CLL cells that was associated with up-regulation of Bcl-2, Bcl-xL,
and Mcl-1 In addition to the herpes virus entry mediator that is a
high affi nity ligand for CD160 it has been shown that classical and
nonclassical MHC class I molecules bind to CD160 with low affi nity
The K562 cell line used to generate the aAPC is characterized as
HLA-Cw3+/Cw5+,with low-level constitutive expression that can be
augmented by IFN-γ To verify if CD160 engagement with MHC class
I is important for NK cell survival in this system we applied w6/32
antibodies to block HLA-C recognition We stained fresh purifi ed NK
cells with CFSE and blocked Fc-receptors with human IgG to prevent
CD16 involvement in NK cell activation In parallel, irradiated
aAPC-mIL21 were incubated with w6/32 antibodies or with isotype-matched control antibodies and after washing cells were co-cultured with NK cells We found that blocking of HLA class I recognition did not affect NK cell proliferation in response to aAPC-mIL21 (fi g.2a) but increased apoptosis (fi g.2b) This data suggests that IL21might prevent activation-induced NK cell death through up-regulation of anti-apoptotic factors through a CD160–mediated mechanism Future experiments will focus on identifying the mechanisms of suppressing apoptosis in this system
High-Capacity, Gutless Adenoviral Vectors in the Nạve Rat Brain: Pre-Clinical Safety and Toxicity Evaluation as a Prelude to a Phase I Clinical Trial for Glioma
A K M G Muhammad,1 Mariana Puntel,1 Weidong Xiong,1
Kyle Kelson,1 Alireza Salem,1 Kurt M Kroeger,1 Chunyan Liu,1
Catherine Farrokhi,1 Liliana Lacayo,1 Robert N Pechnick,1 Donna Palmer,2 Philip Ng,2 Pedro R Lowenstein,1 Maria G Castro.1
1 Cedars-Sinai Medical Center/UCLA, Los Angeles, CA; 2 Baylor College of Medicine, Houston, TX.
Glioblastoma multiforme (GBM) carries a dismal prognosis with
a median survival of 18-21 months post-diagnosis We have recently demonstrated the therapeutic effi cacy and high safety profi le of intratumoral delivery of a novel, combined gene therapy consisting
of fi rst generation, or high-capacity adenoviral vectors (HC-Ads) encoding either the conditionally cytotoxic gene herpes simplex type 1-thymidine kinase (TK) or the immunostimulatory gene fms-like tyrosine kinase 3 ligand (Flt3L) transgenes in a syngeneic, orthotopic rat model of GBM In anticipation of an upcoming dose
fi nding, Phase I clinical trial for primary GBM, we herein performed
a thorough assessment of the safety and toxicity of HC-Ads encoding
TK and Flt3L upon administration into the nạve rat brain Lewis rats were injected intracranially with escalating doses of HC-Ad-TK and HC-Ad-TetON-Flt3L (1x10^8, 1x10^9, or 1x10^10 vp of each) followed by systemic administration of gancyclovir and doxycycline Rats were evaluated at 5 days, 1 month, 6 months and 12 months for biodistribution of HC-Ad vector genomes, HC-Ad vector induced behavioral defi ciencies, neurotoxicity, peripheral blood cell counts and serum biochemistry, circulating levels of Flt3L, anti-adenovirus neutralizing antibodies, and anti-TK antibodies Even at the highest dose tested, real-time quantitative PCR did not detect evidence
of biodistribution of HC-Ad genomes to peripheral organs and a comprehensive panel of behavioral testing did not reveal any vector mediated abnormalities Peripheral blood cell counts and serum biochemistry were within normal ranges during all time points,
at all doses tested Flt3L expression was tightly regulated, with
Trang 2Molecular Therapy Volume 19, Supplement 1, May 2011
CANCER - IMMUNOTHERAPY II
expression dynamics that coincided with the administration of the
inducer doxycycline However, a comprehensive neuropathological
panel revealed evidence of neurotoxicity at the highest dose tested
(1x10^10 vp of each), i.e loss of brain tissue, and high levels of
infl ammation We did not detect any evidence of neurotoxicity or
long-term infl ammation at either lower dose tested Taken together,
these data indicate that 1x10^9 vp of each HC-Ad is the maximally
tolerated dose (MTD) that can be safely administered into the brains
of nạve Lewis rats These well controlled, non-clinical safety and
toxicity studies are required by the FDA in order to fi le of an IND
and constitute a major milestone in the path towards implementation
of the fi rst ever Phase I clinical trial for GBM using gutless, HC-Ad
vectors
Therapy Mediated Brain Tumor Regression
Hikmat H Assi,1,2 James Curtin,1 Chunyan Liu,1 Maria G Castro,1,2
Pedro R Lowenstein.1,2
1 Gene Therapy Research Institute, Cedars Sinai Medical Center,
Los Angeles, CA; 2 Molecular and Medical Pharmacology, UCLA,
Los Angeles, CA.
STAT3 signaling is constitutively active in high-grade tumors and
its transcriptional activity is required by malignant cells for their
proliferation well as promotion of an immunosuppressive tumor
microenvironment Recently pre-clinical models have investigated
whether STAT3 antagonists can be used in combination with the
immunotherapy to induce tumor cell death and enhance immune
responses against GBM antigens Owing to the multiple roles
of STAT3 in both tumor cells and immune cells, we wished to
investigate whether STAT3 signaling is involved in Ad-TK/Ad-Flt3L
gene therapy-mediated T cell dependent brain tumor regression We
used adenovirus expressing STAT3 shRNA as well small molecule
inhibitors to silence STAT3 and assessed its role in tumor cell
proliferation as well as dendritic cell (DC) activity We report that
expression of STAT3 is up regulated in various syngeneic glioma
mouse models Treating Gl26 or SMA560 glioma cells with the novel
small molecule inhibitor CPA-7 blocks STAT3 in vitro and leads to
cell death through a loss of Cyclin D, Bcl-xl and survivin expression
We also show that STAT3 mediates the activation and maturation of
DCs Using bone marrow cells with GM-CSF we generated immature
mouse DCs and examined the effects on DC maturation elicited by
the TLR ligand CpG and the STAT3 inhibitor CPA7 Treatment of
DCs with CPA-7 induced their maturation evidenced by increased
expression of co-stimulatory molecules CD80, CD86 and CD40
as well as decreased antigen uptake indicative of a more mature
phenotype CPA-7 treatment enhanced the maturation activity of
CpG DCs treated with CPA7 in combination with CpG were able
to better stimulate the proliferation of T cells in an allogeneic MLR
when compared to DCs treated with CpG alone We have previously
demonstrated that intra-tumoral delivery of Flt3L and TK using
adenoviral vectors drives T cell dependent tumor regression in
intra-cranial syngeneic glioma (GBM) models in rodents (Ali et al, 2005;
Curtin et al 2009; Candolfi et al, 2009) Intra-tumoral delivery of
STAT3 shRNA, however, completely blocks Flt3L and TK mediated
tumor regression compared with a scrambled non-specifi c shRNA
This was confi rmed using the small molecule inhibitor CPA-7 Our
data suggest that STAT3 signaling is involved in tumor cells’ survival
and DCs’ functions, thus constituting an attractive therapeutic target
for brain cancer
Improved Responses in Immunogene Cancer Treatment over Standard Plasmid Vectors
Patrick F Forde,1 Lindsay J Hall,1 Marcel de Kruijf,1 Gerald C O’Sullivan,1 Declan M Soden.1
1 Cork Cancer Research Centre, University College Cork, Cork, Ireland.
We compared the effi cacy of an enhanced expression vector (EEV) with CMV standard plasmid vector for non viral immunogenetherapy
of solid tumours The EEV contains its own RNA replicase and transcribes mRNA cytoplasmically The transgenes LacZ and GM-CSF/B71 were under the control of the CMV promoter Comparison of the EEV with the standard vector in normal and growing tumours used quantitative RT-PCR, histological tools and in vivo murine tumour growth and survival curves In all normal tissues (liver, spleen, and colon and oesophageal epithelium) there were signifi cant increased RNA/DNA ratios for the EEV over the standard plasmid The EEV gives superior results than standard vector in non viral immunogene therapy of solid growing tumours The EEV was curative in two tumour models and has potential for clinical development Antitumor responses of the EEV vector were both from immune cell recruitment
to the tumour environment and the unrestrained RNA production in the cytoplasm
the Effi cacy of DNA Cancer Vaccine by Activating Dendritic Cells
Chi-Chen Lin,1 Chia-Chiao Shih,1 Ching-Liang Chu.2
1 Institute of Biomedical Sciences, National Chung Hsin University, Taichung, Taiwan; 2 Yeastern Biotechnology Inc, Taipei, Taiwan.
DNA vaccine has been applied in cancer therapy, but the effi cacy remains to be improved The immunostimulatory effect of a fungal immunomodulatory protein Ling Zhi-8 (LZ-8) isolated from
Ganoderma lucidum has been reported on human dendritic cells
(DCs) In this study, we tested the adjuvanticity of LZ-8 for HER-2/ neu DNA vaccine against p185neu expressing tumor MBT-2 in mice
We found that recombinant LZ-8 activated mouse bone marrow-derived DCs via TLR4 and this stimulation was not due to any microbe contaminant In addition, LZ-8 enhanced the ability of DCs to induce
antigen-specifi c T cell activation in vitro and in a subunit vaccine model in vivo Surprisingly, LZ-8 co-treatment strongly improved
the therapeutic effect of DNA vaccine against MBT-2 tumor in mice This increase of anti-tumor activity was attributed to the enhancement
of vaccine-induced Th1 and CTL responses Consistent with the results from DCs, the promoting effect of LZ-8 on DNA vaccine was diminished when the MBT-2 tumor cells were grown in TLR4 mutant mice Thus, we concluded that LZ-8 may be a promising adjuvant to enhance the effi cacy of DNA vaccine by activating DCs
Bi-Specifi c Tumor-Reactive T Cells
Robert C Chan,1 Ceidy Sanchez,1 Anna Worth,1 Ann M Leen,1
Malcolm K Brenner,1 Ganesh Palapattu,2 Juan F Vera.1
1 Baylor College of Medicine, Texas Children’s Hospital, The Methodist Hospital, Houston; 2 Department of Urology, The Methodist Hospital, Houston.
Although localized prostate cancer (PC) can often be cured by surgery and/or radiation therapy, the therapeutic options for castrate-resistant disease (CRPC) are largely palliative, underscoring the need for alternative therapies Like many other tumors, CRPC expresses tumor-associated antigens (TAAs), which are potential targets for immune destruction However, tumors use multiple mechanisms
of immune evasion including downregulation of target antigen