201 A New Epithelial Junction Opener for Cancer Therapy Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S78 CANCER IMMUNOTHERAPY I 199 Phase[.]
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CANCER - IMMUNOTHERAPY I
Tumor Cell – GMCSF/pbi-shRNA™ Furin DNA
Plasmid) Vaccine in Advanced Melanoma
John Nemunaitis,1,2,3,4 Neil Senzer,1,4 Minal Barve,3 Padmasini
Kumar,4 Donald D Rao,4 Gladice Wallraven,4 Beena O Pappen,4
Joseph Kuhn,5 Peter Beitsch,2 Robert Steckler,2 Brian M Gogel,2
Walton Taylor,2 Phillip B Maples.4
1 Mary Crowley Cancer Research Centers, Dallas; 2 Medical City
HCA, Dallas; 3 Texas Oncology, P.A., Dallas; 4 Gradalis, Inc.,
Dallas; 5 WLS Surgical Associates, P.A., Dallas.
The Phase I trial of FANG™ vaccine (Senzer et al Mol Ther 2012;
20:679-686) provided suffi cient safety data to proceed to Phase II
effi cacy studies This decision was bolstered by the correlation of
duration of survival with induced immune response at 3 months
(in patients with serial analysis) Specifi cally, 4 metastatic melanoma
patients in the Phase I trial, who having failed standard therapy had an
expected survival prognosis of 6 months, received a combined total
of 25 vaccinations (i.e 3 patients at dose level 1 x 107 and 1 patient
at 2.5 x 107 cells / injection (database review revealed no evidence
of dose related response)) These patients have thus far experienced a
median survival of 698 days since starting treatment (967, 835, 560,
and 490 days, each) Two of these patients demonstrated induced T
cell activation via IFN gamma ELISPOT assessment by Month 3 post
treatment start One patient did not show positive T cell activation
at the Month 3 ELISPOT assessment but did so by Month 7 One of
the patients who, coming off treatment early and not having a Month
3 assessment, was ELISPOT positive at Month 16 These 4 patients
also showed knockdown of the endogenous immunosuppressive
proteins TGF1 (mean 99% knockdown) and TGF2 (mean 86%
knockdown) mediated by targeted pbi-shRNA™ furin transfected
vaccine product Based on these results, the Phase II trial (CL-PTL
114, BB-IND 14205) was activated following FDA, RAC, IBC and
IRB acceptance Design of the ongoing Phase II study involves an
intradermal injection of the FANG™ vaccine (1.0 x 107 cells/injection/
month; maximum of 12 vaccinations) Four patients with Stage IV
melanoma with biopsy accessible lesions have been treated Neither
hematologic function, liver enzyme, renal function, or electrolyte
assays nor protocol specifi ed physical examinations demonstrated
any toxic effects Immune function analyses, including intratumoral
immune phenotype and ELISPOT analysis of cytotoxic T cell function
to autologous tumor antigens, are ongoing and will be reported Stable
disease has been observed in 2 of 4 treated patients In conclusion,
results support continuation of accrual
Reprogramming of Natural Killer Cells by Soluble
Immune Modulators
Yue Guan,1 Purba Singh,1 Trenae Mann,1 Christopher B
Chambers,1 Donald S Torry,1 Andrew Wilber.1
1 Medical Microbiology, Immunology and Cell Biology, Southern
Illinois University School of Medicine, Springfi eld, IL.
Natural killer (NK) cells classically are associated with immune
surveillance and destruction of tumor cells via cytotoxicity Recent
studies indicate that NK cells with non-classical CD phenotypes
(CD56brightCD16dim-negative) lose their cytotoxic capacity, release
pro-angiogenic factors, and facilitate tumor growth Mechanisms
driving these changes are not established but soluble factors are
thought to direct the unique phenotypic and functional differentiation
of these CD56brightCD16- NK cells We used a combination of
clinical specimens, primary cell culture, and a novel animal model
to evaluate the effects of transforming growth factor beta (TGF)
and prostaglandin E2 (PGE2) on NK cell phenotype and function
patients (N=7) were 92 + 39 pg/mL before surgery and 45 + 11 pg/
mL after nephrectomy (P=0.02, T-test) Post-operative levels of
PGE2 were approaching levels observed for healthy volunteers (27 + 11 pg/mL), suggesting that PGE2 is a RCC tumor-derived factor Phenotype analysis revealed that peripheral blood NK (pNK) cells from healthy volunteers were characteristically CD56dimCD16bright (95%); however, RCC tumor-derived NK cells exhibited the CD56brightCD16- phenotype The tumor-derived NK cells had elevated vascular endothelial growth factor (VEGF) transcripts (3-fold) compared to freshly isolated pNK cells Trans-differentiation
of CD56dimCD16bright pNK cells to the CD56brightCD16dim phenotype was achieved after culture with TGF; this conversion was coincident with impaired NK cytotoxicity (>50% reduction) and
a modest augmentation of VEGF mRNA upon crosslinking of the activating receptor NKp46 A Balb/c mouse renal adenocarcinoma cell line (Renca) expressing moderate levels of TGF (300-350 pg/mL) was engineered for PGE2 expression with murine Cox2 in antisense (Cox2-) or sense (Cox2+) orientations ELISA demonstrated that Cox2- cells produced background levels of PGE2 (35 pg/mL) while Cox2+ cells produced >5,000 pg/mL PGE2 Exposure of freshly isolated human pNK cells to conditioned supernatants from Cox2- (TGF only) or Cox2+ (TGF and PGE2) cells reduced cytotoxicity by 20% and 30%, respectively In vivo studies demonstrated rapid tumor growth and metastasis over three weeks following kidney capsule injection in Balb/c mice Lymphocytes isolated from these RCC-like tumors were infi ltrated with NK-cells (CD3-p46+), nearly 60% of which demonstrated diminution or absence of CD16 Collectively, these studies support a role for TGF and PGE2 in trans-differentiation
of CD56dimCD16bright pNK cells to CD56brightCD16dim-negative cells which have lost their normal destructive capacities and, instead, support tumor growth and metastasis Studies designed to inhibit the immune suppressive functions of these molecules are planned and may provide new therapeutic avenues for future cancer treatments
201 A New Epithelial Junction Opener for Cancer Therapy
Ines Beyer,1 Hua Cao,1 Jonas Persson,1 Roma Yumul,1 Andre Lieber.1
1 Medicine, University of Washington, Seattle.
Most solid tumors are of epithelial origin Epithelial cancers maintain intercellular junctions that link cancer cells together and prevent the penetration of therapeutics deep into the cancer Several studies demonstrated that the upregulation of epithelial junction proteins correlated with increased resistance to therapy, including therapy with monoclonal antibodies and chemotherapeutics One of the epithelial junction proteins is desmoglein 2 (DSG2) Recently,
we demonstrated that a group of human adenoviruses (Ad) (Ad serotype 3, 7, 11, and 14) use DSG2 as a primary attachment receptor for the infection of cells DSG2 is overexpressed in epithelial malignancies We have created a small recombinant protein derived from Ad serotype 3 This protein binds to DSG2 and triggers transient opening of epithelial junctions We therefore named the protein
“JO-1” (“junction opener -1”) JO-1 is produced in E.coli and can
be easily purifi ed In a number of xenograft tumor models, we have shown that intravenous injection of JO-1 increased the effi cacy of monoclonal antibody (e.g trastuzumab/Herceptin and cetuximab/ Erbitux) therapy Many chemotherapy drugs are larger than 500
Da, the upper limit of molecules that are able to pass through tight junctions We have demonstrated that the therapeutic effects of several of these drugs, including paclitaxel, nab-paclitaxel, docetaxel, irinotecan, and liposomal doxorubicin are increased by co-therapy with JO-1 Furthermore, we have shown that JO-1 leads to massive accumulation of chemotherapy drugs in the tumor, decreasing the drug concentrations in the rest of the body This resulted in the elimination
of chemotherapy-related toxicities to bone marrow, liver, and intestine Toxicology studies carried out in mice and monkeys have
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CANCER - IMMUNOTHERAPY I
shown that intravenous injection of JO-1 is safe and well-tolerated
Additional in vivo studies have demonstrated that JO-1 remains active
in the presence of anti-JO-1 antibodies In immunocompetent DSG2
transgenic mice with syngeneic tumors, JO-1 signifi cantly enhanced
PEGylated liposomal doxorubicin (Doxil) therapy when given in
multiple cycles with an interval of 2 weeks More recently, we have
used immunocompetent animal models to demonstrate that JO-1
treatment also enhances the intratumoral penetration and effi cacy of
anti-tumor T-cells We are now focused on the clinical translation of
JO-1 in combination with Doxil for the treatment of ovarian cancer
Expansion of Transgenic T Cells
David Rushworth,1 Laurence Cooper.1
1 Pediatrics, MD Anderson Cancer Center, Houston, TX.
Adoptive transfer of T cells as a therapy for cancer is developing
rapidly, and the use of transgenic T cells has shown promise in
early clinical trials for multiple purposes related to cancer therapy
However, these therapies have severe side effects and high costs
related to conditioning the patient and infusing enough T cells into the
patient to stably engraft cancer-specifi c T cells We have developed a
novel method to selectively grow T cells in the presence of cytotoxic
chemotherapeutics These chemotherapy resistant T cells provide
an opportunity for novel T cell expansion and infusion regimens
that could treat cancer with chemotherapeutic agents as well as
chemotherapy resistant T cells potentially decreasing the cost and time
required to effectively treat patients with T cells Here we describe
the unique properties of transgenically modifi ed methotrexate and
5-fl uorouracil resistant T cells that have also been redirected to target
B cell leukemia antigen CD19 using a chimeric antigen receptor
(FANG™) Vaccine, Incorporating Bifunctional
shRNAfurin and GMCSF Transgene Expression, in
Advanced Ewing’s Sarcoma: Preliminary Data in
Pediatrics Patients
Maurizio Ghisoli,1 Carl Lenarsky,1 Gladice Wallraven,2 Padmasini
Kumar,2 Phillip B Maples,2 John Nemunaitis.1,2,3,4
1 Texas Oncology, P.A, Dallas; 2 Gradalis, Inc., Dallas; 3 Mary
Crowley Cancer Research Centers, Dallas; 4 Medical City HCA,
Dallas.
The FANG™ vaccine contains a plasmid encoding both GMCSF
and an RNA interference (RNAi) moiety, bifunctional shRNAfurin
(pbi-shRNA™ furin), that targets and down regulates the proprotein
convertase Furin resulting in knockdown of both TGFb1 and b2
The pbi-shRNA™ technology is designed to maximize target
inhibition via translational repression through a process involving
simultaneous mRNA cleavage and p-body sequestration GMCSF
further activates and expands the cytotoxic T-cell population in the
immune afferent arm This “triad” concept promotes immune
de-tolerization, enhances antigen presentation and antigen processing
Based on initial encouraging results in adult cancer patients at Mary
Crowley Cancer Research Center with mechanistic demonstration of
the technology (mean Furin knockdown was 85.09% and mean TGFb1
and -b2 knockdown were 93.5% and 92.5%, respectively), survival
advantage (median survival was 554 days, FANG™ versus 255 days,
non FANG™, (p=.006)) and with the lack of serious adverse events,
a Phase I study was amended to include pediatric patients ( 12 y.o.)
with advanced/relapsed Ewing’s sarcoma who had failed standard
treatment options Briefl y, the vaccine is cGMP manufactured at
Gradalis, Inc following autologous tumor tissue harvest Vaccine is
produced at a dose of 1 x cells/injection The vaccine is administered
intradermally once a month (minimum of 5 to a maximum of 12
doses) ELISPOT analysis of cytotoxic T cell function to autologous
tumor antigens are monitored at baseline, Months 2, 4, 6 and EOT
To date, 4 pediatric patients entered the screening process for entry into trial, 3 with relapsed Ewing’s Sarcoma and 1 with progressive disease Vaccine manufacturing was successful in 3 of the 4 harvested patients, 1 patient had insuffi cient viable tumor tissue Two patients were registered into the protocol, one died from progressive disease (received one FANG™ vaccine injection) One patient who developed pulmonary metastasis one year post completion of treatment has stable disease 6 month after treatment initiation and showed activation of the ELISPOT assay There have been no treatment-related serious adverse effects; one patient had a grade 1 treatment-related AEs limited to the skin erythema at the injection site This limited initial data shows the safety and feasibility of this study Updated results will be presented
Repertoire in Different Immunological Conditions
by Deep Sequencing
Eliana Ruggiero,1 Jan P Nicolay,2,3 Anne Arens,1 Raffaele Fronza,1 Anna Paruzynski,1 Ali Nowrouzi,1 Gökçe Ürenden,1 Sergij Goerdt,3 Hanno Glimm,1 Peter H Krammer,2 Manfred Schmidt,1 Christof von Kalle.1
1 Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany; 2 Division of Immunogenetics, German Cancer Research Center, Heidelberg, Germany; 3 Department of Dermatology, Venereology and Allergology, University Medical Center, Mannheim, Germany.
T cell ability to recognize foreign antigens relies on the high variety
of T cell receptors (TCR), heterodimeric or glycoproteins Each chain is originated from the somatic rearrangement of non contiguous genes belonging to different families (V, (D, only for
and chains) and J) Addition and deletion of non templated nucleotides at the rearranging loci further increase the diversity of the TCR molecule By deep sequencing of the CDR3 region, the hypervariable portion of the TCR responsible for antigen recognition,
it is possible to identify the clonality and the functional status of a T cell population We have established TCR-LAM direct sequencing, a new unbiased, highly specifi c and sensitive RNA-based assay for the characterization of the TCR repertoire diversity in healthy and in diseased conditions Analysis of the TCR repertoire in 3 healthy HLA-unrelated individuals led to the dissection of the events generating the TCR molecules A not uniform V and J gene usage and the occurrence
of preferred VJ pairings were observed A deep insight in the CDR3 sequence down to the single nucleotide level, displayed a high degree
of variability in the center of the sequence with a preferential usage
of guanines These results hold true for both TCR chains and for every donor Convergent recombination, the generation of identical TCR amino acid (aa) specifi cities through different VJ pairings and/
or different nucleotide sequences, was observed in 11% and 7%
chain CDR3 aa sequences A sharing of 2.4% and 1% TCR
aa sequences between any 2 donors (public clones) was identifi ed in our dataset Sequencing results from a blinded analysis of the TCR repertoire in 10 patients with severe and mild form of cutaneous T cell lymphoma (Sézary disease) showed the ability of TCR-LAM
to dissect different stages of the disease We observed a distorted oligoclonal repertoire with a dominating TCR clonotype in the severe disease form and a less restricted oligoclonal repertoire in the mild disease form Moreover, imunomonitoring of the TCR diversity was performed on peripheral blood mononuclear cells (PBMC) in 2 CMV seropositive donors after restimulation of the CMV-specifi c T cell response with a viral peptide pool Overtime (day 0, day 9 and day 15 after restimulation) analysis showed the emergence of specifi c TCR clonotypes already at day 9 CMV-specifi c sequences were detected
at day 0 even with a very low frequency (0.01%) A high degree of convergent recombination and the presence of TCR motifs, showed