Abstracts for the 25th Annual Scientific Meeting of the International ...

J Immunother Volume 33, Number 8, October 2010
Abstracts
with a single peptide pool for 2 hours, and recombined for another
4 hours. The IFN-g Secretion Assay was used to magnetically
enrich IFN-g secreting T cells to a purity of >90%. High antigen
specificity and functionality of the enriched T cells was confirmed
after expansion for 9 to 14 days. T cell lines contained high
frequencies of pp65tetramer+ CD8+ T cells. Additionally, after
restimulation with a mixture of peptide pools 21% to 53% of
CD4+ T cells and 53% to 87% of CD8+ T cells produced IFN-g.
Moreover, comparing T cell frequencies specific for each single
antigen in PBMC with that in multivirus-specific T cell line after
separate reactivation with peptide pools by analysis of IFN-g
production, we observed the specificity for each antigen sustained
after enrichment and expansion. We found similar results in
number of enriched cells, expansion rates, and antigen-specificity of
the T cell lines, regardless of whether PBMC loaded with a mixture
of antigens or separately with single peptide pools to generate T cell
lines. In summary, we established a protocol for rapid in vitro
generation of multiantigen-specific CD4+ and CD8+ T cells using
a combination of peptide pools from several antigens for
restimulation and subsequent magnetic selection of IFN-g secreting
T cells.
Hypotheses for Improving Designer T Cells; Phase I Trials
Richard Junghans*w, Ritesh Rathore*w, Barti Rathore*w,
Qiangzhong Ma*w, Anthony Bais*, Erica Gomes*, Ryan
Harvey*, Nithiandan Selliah*w, Shah Miah*w, Pam Davol*,
Steven Cohen*, Samer Al Homsi*w. *Surgery, Roger Williams
Hospital, Providence, RI; w Boston University School of Medicine,
Boston, MA.
In prior Phase I trials of 1st generation (1st gen) designer T cells,
proof-of-principle ‘‘biologic responses’’ were noted, but with poor
in vivo persistence and transient in-tumor activity. The first
problem generated the hypothesis: If T cells were maintained
systemically at high levels, a sustained T cell percolation into
tumor could yield cures even if T cells survived for only a few
days of killing. The second problem generated the hypothesis: If
T cells entering tumor, though few in number, were to proliferate
on contact with antigen, then tumors could be cured. In
separate clinical trials, these hypotheses are being addressed in our
group.
Methods: Patient T cells are transduced and expanded ex vivo to
span dose levels of 10 4 9, 10 4 10 and 10 4 11 T cells. The first study
uses 1st gen designer T cells (against prostate specific membrane
antigen, PSMA), but with prior non-myeloablative (NMA)
conditioning to create a ‘‘hematopoietic space’’ into which designer
T cells are engrafted for prolonged in vivo persistence, with coadministered
continuous infusion IL2. The second study uses a
simple infusion protocol that applies 2nd gen designer T cells
(against CEA) with added CD28 co-stimulation with the goal of in
situ amplifying the effector cells that reach tumor.
Results: Each study treated five patients to date at the low 10^9 and
middle 10 4 10 T cell doses. NMA conditioning led to successful
T cell engraftment in the 1% to 20% range at one month, versus
designer T cells being undetectable at the same time in the infusion
study. No on-target T cell toxicities were noted in either study. In
the engraftment protocol, two patients had PSA reductions of 50
and 70% in the two months following treatment. In the second
study with 2nd gen anti-CEA T cells, one gastric cancer patient had
shrinkage of lung and brain mets and another has stable disease for
12+ months after treatment; future patients will have IL2 added
with the next T cell dose escalation. The clinical (non-manufacturing)
cost per patient was estimated at $60 to 100 K in the
engraftment study and $5 to 10 K in the infusion study.
Conclusion: Parallel approaches are being applied to test hypotheses
of benefit of engraftment versus benefit of co-stimulation to
create effective anti-tumor activity in designer T cells. Study results
support the safety of the protocols with indications of efficacy but
the dose escalations are at an early stage. Patient recruitments are
continuing.
Functional Reprogramming of the Tumor Stroma by IL-12
Engineered T Cells is Required for Anti-tumor Immunity
Sid P. Kerkar*, Robert Reger*, Pawel Muranski*, Zhiya Yu*,
Douglas Palmer*, Dhanalakshmi Chinnasamy*, Christopher
A. Klebanoff*, Yun Ji*, Luca Gattinoni*, Steven A. Rosenberg*,
Giorgio Trinchieriw, Nicholas P. Restifo*. *Center for Cancer
Research; w Cancer and Inflammation Program, National Cancer
Institute, Bethesda, MD.
Bone marrow derived stromal cells within the tumor microenvironment
are capable of cross presenting antigens to cytotoxic
T lymphocytes (CTL). We found that the adoptive transfer of
tumor-specific CD8+ T cells gene-engineered to secrete IL-12 led
to the increased local infiltration of adoptively transferred T cells
and caused the regression of large established B16 melanomas. The
autocrine effects of IL-12 resulted in the production of large
amounts of IFN-g by T cells. Surprisingly, we found that IL-12-
engineered T cells that lacked the ability to receive signals from
IL-12 (Il12rb2 / ), and indeed T cells that lacked the ability to
produce IFN-g (Ifng / ), retained all of their ability to trigger
tumor destruction. However, tumor treatment efficacy was
abrogated when the cells in host mice lacked IL-12 receptors
(Il12rb2 / ), IFN-g receptors (IfngR / ) or the ability to
produce IFN-g. Thus, sensitization of host cells and not the
transferred T cells within the tumor microenvironment was critical
for successful anti-tumor immunity. We measured increased
endogenous CD8+ and host NK cells within the tumor but
treatment responses remained robust in mice completely devoid of
T and B cells (Rag / ) and depleted of NK cells. We found that
the majority of cells expressing the IL-12Rb2 receptor within the
tumor were CD11b+ myeloid cells. Transfer of IL-12-producing
anti-tumor T cells triggered significant in situ changes in CD11b+
cells including increased expression of H-2Db along with upregulation
of Fas (CD95), and FADD. In addition, both the
numbers and percentages of CD11b+ cells dropped just prior to
tumor regression. Tumor treatment was abrogated in mice deficient
in MHC class I (b2M / ) but not class II (I-Ab / ),
indicating the functional importance of antigen cross-presentation
in vivo. These results are consistent with a model whereby IL-12
triggers the functional maturation of in situ APCs capable of crosspresenting
tumor antigens. Licensed recognition of these antigens
by tumor-specific T cells may in turn trigger the collapse of the
tumor stroma and its vasculature.
Monomeric Designer T Cells Kill IL13Ra2 Expressing GBM
More Efficiently
Seogkyoung Kong, Richard P. Junghans, Prakash Sampath.
Neurosurgery, Boston University School of Medicine, Roger
Williams Medical Center, Providence, RI.
GBM is a devastating primary brain tumor for which there is no
effective therapy to specifically target tumor cells. Targeted
immuno-gene therapy has been shown effective in a number of
tumor models without significant toxicity. We aim to develop
potent GBM-specific T cells as an innovative and unique targeted
immuno-gene therapy. I exploit IL13Ra2 as a GBM-specific tumor
antigen due to its frequent overexpression on a majority of GBM
but not on normal brain tissues. Targeting IL13Ra2 on GBM has
a strong rationale supported by clinical development of IL13
immunotoxin molecules. IL13 binds two types of receptor with
different affinities: IL13 binds to GBM-associated IL13Ra2 with
high affinity (Kd = 0.25 to 1.2 nM); IL13 binds to IL13Ra1 first
with low affinity (Kd = 2 to 30 nM) and then recruits IL4Ra to the
complex. Previous studies showed that substitutions at three sites
of IL13 (Glu-11, Lys-103, or Arg-107) proved to be critical for
IL13 binding to IL13Ra2, and modifications of these sites can
neutralize or dramatically increase the affinity of IL13 to IL13Ra2,
compared of the shared IL13Ra1, which is expressed on the normal
brain cells and other tissues. In order to achieve both specific
targeting and selectively enhanced cytotoxicity of GBM-associated
r 2010 Lippincott Williams & Wilkins www.immunotherapy-journal.com | 861