11 AAV Mediated CNS Gene Transfer of Bevacizumab Reduces Human Glioblastoma Growth and Increases Survival in Mice Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society o[.]
Trang 1Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
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CANCER - IMMUNOTHERAPY I
evasion from immunological control including apoptosis resistance,
insensitivity to anti-growth signals, downregulation of MHC, NK
cell evasion, immune suppression among others Consequently, it is
essential to develop innovative cancer strategies to address immune
obstacles to generate more effective immune therapies
Myeloid-derived suppressor cells (MDSCs) are a mixture of immune cells
composed of immature macrophages, granulocytes, dendritic cells,
and myeloid cells These cells possess a remarkable ability to suppress
immune responses, and as part of tumor immune suppression MDSCs
are able to expand, facilitating tumor growth, and immune escape
These cells represent an important area of research as MDSCs may
serve as a target for preventing tumor progression and could affect
immune responses to anti-cancer vaccines In mice, we investigated
the mechanisms of MDSCs expansion and characterized the role of
MDSCs in T cell immune suppression in an antigen specifi c B16
melanoma tumor model utilizing a novel Tyrosinase DNA vaccine
Using this potent cellular response-driving Tyr-DNA vaccine with
electroporation (EP), we showed the immunogenicity of Tyr vaccine
as a prophylactic measure The vaccine increased the magnitude
and broadened the immune response by strengthening CD8+ T-cell
infi ltration at the tumor site in part by blocking MDSCs expansion
This blocking was through regulating a potent angiogenic factor for
MDSC expansion, MCP-1 production Combining DNA vaccines
with blocking of MCP-1 production may prove important for
treatment of Melanoma by immune therapy Further studies in this
area are warranted
10 First-in-Human Application of Sleeping
Beauty System for Gene Therapy
Partow Kebriaei,1 Helen Huls,2 Harjeet Singh,2 Simon Olivares,2
Matthew J Figliola,2 Margaret Dawson,2 Bipulendu Jena,2 Rineka
Jackson,2 Doyle Bosque,1 Ian McNiece,1 Gabriela Rondon,1 Perry
B Hackett,3 Elizabeth J Shpall,1 Richard E Champlin,1 Laurence
J N Cooper.2
1 Stem Cell Transplantation and Cellular Therapy, MD Anderson
Cancer Center, Houston, TX; 2 Pediatrics, MD Anderson Cancer
Center, Houston, TX; 3 Genetics, Cell Biology and Development,
University of Minnesota, Minneapolis, MN.
T cells can be genetically modifi ed ex vivo to redirect specifi city
upon enforced expression of a chimeric antigen receptor (CAR)
that recognizes tumor-associated antigen (TAA) independent of
human leukocyte antigen (HLA) We report a new approach to
non-viral gene transfer using the Sleeping Beauty (SB) transposon/
transposase system to stably express a 2nd generation CD19-specifi c
CAR (designated CD19RCD28 that signals through chimeric
CD28/CD3-) in autologous and allogeneic T cells manufactured in
compliance with current good manufacturing practice (cGMP) for
Phase I/II trials T cells were electroporated using a Nucleofector
device to synchronously introduce plasmids carrying an SB
transposon (CD19RCD28) and encoding a hyperactive SB transposase
(SB11) T cells stably expressing the CAR were retrieved over 28
days of co-culture by recursive additions of g-irradiated artifi cial
antigen presenting cells (aAPC) in presence of soluble recombinant
interleukin (IL)-2 and IL-21 The aAPC (designated clone #4) were
derived from K562 cells and genetically modifi ed to co-express the
TAA CD19 as well as the co-stimulatory molecules CD86, CD137L
and a membrane-bound protein of IL-15 Using the dual platforms
(Figure) of SB system and aAPC to date we have enrolled patients
with multiple relapsed ALL (n=4) or B-cell lymphoma (n=5) on
three investigator-initiated trials to infuse thawed patient- and
donor-derived CD19-specifi c T cells after autologous (n=2), and
allogeneic adult (n=6) and umbilical cord (n=1) hematopoietic
stem-cell transplantation (HSCT) Each T-stem-cell product was subjected to a
battery of in-process testing to complement release testing The fi rst
research participant was successfully treated in May of 2012 We have
administered 5 infusions to date in patients receiving adult allogeneic HSCT, beginning at a dose of 106 and escalating to 5x107 T cells/m2
No toxicities have been noted These HSCT trials continue to accrue and pave the way for our next trials infusing autologous CAR+ T cells (including T cells activated via chimeric CD137/CD3-) after lympho-depleting chemotherapy We note that the purifi ed plasmids can be produced in bulk at a fraction of the cost of clinical grade virus, which enhances the translational appeal of electroporation and propagation of T cells in compliance with cGMP This is the fi rst report of the fi rst human application of the SB system to genetically modify clinical-grade cells and provides investigators with a new approach to gene therapy
Bevacizumab Reduces Human Glioblastoma Growth and Increases Survival in Mice
Martin J Hicks,1 Kosuke Funato,3 Lan Wang,1 Eric Aronowitz,2
Jonathan P Dyke,2 Douglas J Ballon,2 Vivianne S Tabar,3 David
F Havlicek,1 Esther Z Frenk,1 Bishnu P De,1 Maria J Chiuchiolo,1
Dolan Sondhi,1 Neil R Hackett,1 Stephen M Kaminsky,1 Ronald
G Crystal.1
1 Department of Genetic Medicine, Weill Cornell Medical College, New York, NY; 2 Department of Radiology, Weill Cornell Medical College, New York, NY; 3 Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY.
Glioblastoma multiforme (GBM) accounts for approximately 54% of all primary brain and CNS gliomas It is distinguished by prominent vascular proliferation, and rapid and invasive growth Despite increases in the molecular and physiological characterization
of GBM, the median overall survival of GBM patients remains little more than one year Current therapies to reduce disease progression are typically administered systemically and thus, are limited by the blood-brain barrier We have developed a method to deliver the genetic message for therapeutic monoclonal antibodies (mAB) directly to the CNS via adeno-associated virus (AAV) gene transfer vectors such that expression is persistent and local to the milieu of the tumor To measure the effi cacy of this strategy, either the U87MG cell line
or patient-derived early passage GBM cells were administered to the striatum of NOD/SCID immunodefi cient mice Quantifi cation
of GBM tumors was assessed by MRI and immunohistochemical analyses AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin®), an anti-human vascular endothelial growth factor (VEGF) monoclonal, was delivered to the area of the GBM xenograft, either 1 wk post-xenograft administration or simultaneous with xenograft administration Localized expression of the mAB was confi rmed by ELISA, and Western and immunohistochemical analyses Administration of AAVrh.10BevMab to the CNS 1 wk after GBM xenograft reduced the growth (as measured by MRI) of the U87MG tumor by 3.6-fold at 4 wk (p<0.03) with a 64% increase in
survival (treated 54 ± 4 days vs non-treated, 33 ± 3 days, p<0.004),
whereas concurrent administration of the U87MG tumor and AAVrh.10BevMab reduced tumor growth by 5-fold at 3.5 wk (p<0.03)
with a 42% increase in survival (treated 47 ± 3 days vs non-treated,
33 ± 2 days, p<0.01) and reduced tumor blood vessel density (treated
1.1 ± 0.1 % vs non-treated, 3.9 ± 1.0 %, p<0.05) Similar studies of
AAVrh.10BevMab concurrently administered with patient-derived early passage GBM primary cells showed a reduction in primary tumor burden by 3.3-fold at 12 wk (p<0.04) with increased survival
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CANCER - IMMUNOTHERAPY I
(treated 90% alive vs non-treated, 52% alive at 12 wk, p<0.006)
This study shows the effi cacy of AAV-mediated gene transfer of
an anti-angiogenesis therapy to reduce tumor burden and increase
survival in mice with standard laboratory and patient-derived GBM
tumors AAV-mediated gene transfer directly to the CNS overcomes
a common hurdle to anti-tumor therapy, the blood-brain barrier, and
through local and persistent delivery, may provide effective treatment
of glioblastoma
Immunotherapy with Chimeric Antigen Receptors
Andras Heczey,1 Daofeng Liu,1 Amy Courtney,1 Ekaterina
Marinova,1 Gengwen Tian,1 Eric Yvon,2 Jie Wei,1 Gianpietro Dotti,1
Leonid Metelitsa.1
1 Center for Cell and Gene Therapy, Baylor College of Medicine,
Houston, TX; 2 M D Anderson Cancer Center, Houston, TX.
Advances in the design of chimeric antigen receptors (CARs)
improved functionality and therapeutic effi cacy of redirected T cells
in early-phase cancer clinical trials However, high heterogeneity
of CAR-carrying T cells limits their anti-tumor potential and is
associated with increased risk of toxicity We proposed that CAR
expression in CD1d-reactive V24-invariant Natural Killer T-cells
(NKTs) could build upon natural anti-tumor properties of these cells
while restriction by monomorphic CD1d limits their toxicity To test
this prediction we ex vivo expanded and genetically modifi ed primary
human NKTs to express a CAR against GD2 ganglioside, which is
highly expressed in neuroblastoma We compared CAR constructs
that encoded CD3 chain alone (Gz) or with CD28 (G28z), 4-1BB
(GBBz), or CD28 and 4-1BB (G28BBz) co-stimulatory endodomains
Upon retroviral transduction, 50-70% NKTs expressed CAR on the
cell surface and the expression remained stable for at least 3 weeks
after transduction Regardless of the construct composition, CAR
expression rendered NKTs highly cytotoxic against GD2-positive
neuroblastoma cells without affecting their native CD1d-dependent
reactivity The analysis of NKT-cell cytokine profi le in response to
CAR activation revealed that constructs that contained the 4-1BB
endodomain (GBBz or G28BBz) produced a striking Th1-like
polarization of NKTs that was associated with 5-fold amplifi cation
of IFN and GM-CSF and concomitant 2- and 6-fold reduction of
IL-4 and IL-10 production compared with Gz (P<0.001, t-test)
This 4-1BB-mediated NKT-cell polarization was associated with
activation of MAPK and NF-B signaling pathways and more than
10-fold increase in EGR1 mRNA expression within one hour after
CAR binding to GD2-expressing neuroblastoma cells In contrast,
Gz or G28z induced little or no EGR1 up-regulation G28BBz CAR
NKTs had potent and long-lasting anti-tumor activity in a metastatic
model of neuroblastoma in humanized NOD/SCID/IL-2(null) mice
that prolonged animal survival after a single injection of 5X106 cells
(P<0.005, Log rank test) Furthermore, in contrast to CAR T cells
from the same donors, CAR NKTs did not induce multi-organ
graft-versus-host disease These results establish the potential of NKTs to
serve as a safe and effective cellular platform for anti-tumor CAR
therapy that can be used in combination with autologous or allogenic
stem cell transplantation
Tumor Improves T-Cell Therapy
Hua Cao,1 Ines Beyer,1 Vy Phan-Lai,1 Nora Disis,1 Andre Lieber.1
1 Medicine, University of Washington, Seattle, WA.
Extracellular matrix (ECM) and epithelial junctions in solid
tumors affect the effectiveness of therapeutics through blocking of
intratumoral diffusion and/or physical masking of target receptors
on malignant cells We tested whether the removal of these physical
obstacles would facilitates pre-existing and transplanted anti-tumor
T-cells to control tumor growth The fi rst approach was aimed on transient degradation of ECM proteins by the peptide hormone relaxin after stem cell mediated gene transfer In a breast cancer model, we
demonstrated that relaxin augmented the effi cacy of neu-targeted
adoptively transferred T cells, and improved survival of mice with
neu-expressing mammary tumors At day 33, in the T-cell transplanted
group, 25% mice were alive Combined with Relaxin expression survival increased to 62.5% Relaxin expression combined with nạve Tc treatment also increased survival (37.5%), compared to nạve Tc treatment alone (0%) Better survival of relaxin expressing
mice was due to a higher number of neu-specifi c T-cells inside the
tumor The second approach was focused on the transient opening
of epithelial junctions using a recombinant protein (JO-1) that binds
to desmoglein 2 (DSG2) and triggers pathways reminiscent of an epithelial-to-mesenchymal transition in malignant epithelial cells
In this approach, we used TC1 cells that expressed DSG2 TC1 cells also express HPV E6 and E7 and upon injection into C57Bl6 mice trigger E7-specifi c T-cells These tumor-specifi c T-cells are however not able to control tumor growth and 100% of tumor-bearing animals reach the survival end point by day 40 Two injections of JO-1 into tumor-bearing mice at day 7 and 14 resulted in tumor regression in 30% of mice This effect was due to an increased number E7-T-cells inside the tumor and could be blocked by systemic depletion of CD8 cells We are currently testing a combination of JO-1 with systemic depletion of regulatory T-cells
Receptor Modifi ed, Tri-Virus Specifi c Cytotoxic T Lymphocytes after HLA Mismatched Allogeneic Stem Cell Transplantation for Relapsed, Refractory Neuroblastoma
Doug Myers,1 Jiali Sun,2 Cliona M Rooney,2 Natalia Lapteva,2
Adrian P Gee,2 Gianpietro Dotti,2 Bambi Grilley,2 Malcolm K Brenner,2 Robin Ryan.1
1 Pediatric Hematology/Oncology, Children’s Mercy Hospital, Kansas City, MO; 2 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX.
Relapsed/refractory neuroblastoma (rNB) has a dismal prognosis Encouraging recent results using chimeric antigen receptors (CAR) targeting B cell malignancy and neuroblastoma lead us to develop
a study of trivirus specifi c cytotoxic T lymphocytes (CTL) for use
in patients who have received allogeneic stem cell transplantation (SCT) for relapsed resistant neuroblastoma By modifying virus specifi c CTL to express a neuroblastoma targeting CAR (GD2) we hope to obtain; 1) protection against three viruses common after SCT; 2) freedom from GvHD, since virus specifi c CTLs should not
be alloreactive; 3) anti-tumor activity mediated by the GD2 CAR and augmented by the costimulatory signals received by the virus specifi c CTLs when their native receptors engage viral antigens on antigen presenting cells We now report the details of the study and results from the fi rst patient treated Patient 001 is an 11year old male with relapsed neuroblastoma At transplant, he had extensive disease
in his long bones, pleura, skull, and orbit and 100% replacement of marrow with tumor on biopsy His HVA (homovanillic acid) level, a marker of tumor activity, was 131 (ULN 12.8) He received an HLA haploidentical transplant from his mother following conditioning with Campath, fl udarabine, Melphalan and 400cGy TBI He received 8.3x10e6/kg CD34+ cells containing <2x10e4/kg CD3+ cells Engraftment of neutrophils occurred on Day+11, and FISH for XY showed 100% donor chimerism at day +28 On day 43 he received GD2-CAR CTL (1x10e7 CTL/m2) By day +13 after infusion HVA was 11.2 (nml) versus 13 immediately pre-infusion Since infusion, bone pain requiring high dose narcotics and wheelchair assistance has fully resolved LDH has fallen from 2869units (ULN 840units)
to 581 In contrast to previous studies of autologous GD2-CAR