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

J Immunother Volume 33, Number 8, October 2010
Abstracts
responses through the capacity to degrade the essential amino-acid
tryptophan into kynurenine and other downstream metabolites
that suppress effector T cell function and favour the differentiation
of regulatory T cells. IDO can be expressed by a variety of cell
types, including dendritic cells (DC), tumor cells and stromal cells.
IDO is widely dysregulated in tumors and tumor-draining lymph
nodes, where it can mediate immune tolerance to tumor antigens
and facilitate immune escape. Recently, we showed that IDO itself
is subject to cellular immune responses. Hence, spontaneous
cytotoxic T cell reactivity against IDO is present in peripheral
blood as well as in the tumor microenvironment of different cancer
patients. We demonstrated that these IDO reactive T cells are
indeed peptide specific, cytotoxic effector cells. Hence, IDO reactive
T cells are able to recognize and kill tumor cells including directly
isolated AML blasts as well as IDO-expressing dendritic cells, that
is, one of the major immune suppressive cell populations.
Consequently, IDO may serve as an important and widely
applicable target for anti-cancer immunotherapeutic strategies.
Here, we describe that spontaneous cytotoxic T cell reactivity
against IDO also exists in healthy individuals. We show that such
IDO-specific T cells boost immunity against viral antigens by
eliminating IDO+ suppressive cells. This has profound effects on
the balance between IL-17-producing CD4+ T cells and regulatory
T cells. Furthermore, IDO-specific T cells can be induced in healthy
individuals by non-specific inflammatory stimuli such as IFN-g and
IL-2. In the clinical setting, IDO may serve as an important and
widely applicable target for immunotherapeutic strategies where
IDO constitute a significant counter-regulatory mechanism induced
by pro-inflammatory signals. Regulatory T cells have so far been
defined as a specialized subpopulation of T cells that act as
suppressor T cells. However, here we describe effector T cells with a
general regulatory function that may play a vital role for the
mounting or keeping of an effective adaptive immune response.
Pentostatin Plus Cyclophosphamide to Eliminate Immunogenicity
of Anti-Mesothelin Immunotoxin (SS1P)
Justin Taylor, Miriam E. Mossoba, Paul R. Massey, Masanori
Onda, Ira Pastan, Daniel H. Fowler. National Cancer Institute,
Bethesda, MD.
The immunotoxin (IT) SS1P is composed of an anti-mesothelin
Fv fused to a 38 kDa fragment of Pseudomonas exotoxin A and is
being evaluated in mesothelioma and lung cancer. But in
mesothelioma patients, neutralizing antibodies develop after 1
cycle of therapy, thereby preventing additional treatment cycles. IT
therapy has been shown to be effective in hairy cell leukemia, where
multiple cycles can be given without neutralizing antibody
formation. This lack of immunogenicity may result from immune
dysfunction, either from the disorder itself or from previous
immune depleting therapy. In this study, our aim was to determine
if using chemotherapy to dysregulate the immune system,
specifically comparing immune depletion to immune suppression,
might abrogate immunogenicity. BALB/c mice were injected with
10 mg SS1P weekly for 3 weeks and serum was evaluated for
neutralizing antibodies at the end of each week. An immune
depletion group was treated for six days prior to the first IT
immunization with daily cyclophosphamide (50 mg/kg/d) and
pentostatin (1 mg/kg/d) every other day, followed by weekly
maintenance doses of P/C one day prior to immunization. An
immune suppression group was treated on an every other day
schedule of rapamycin (3 mg/kg/d) for the entire post-immunization
period. Three weeks after the first immunization spleen cells
were isolated, counted and analyzed by flow cytometry to
determine the levels of depletion of CD4+ and CD8+ T cells,
B cells (B220+), and myeloid cells (Gr-1+). Immunogenicity was
determined by presence of antibodies to SS1P by ELISA (DC) or
conformational ELISA (ICC) assays. Both the regimens of P/C and
R were safe and well tolerated in the mice in terms of weight loss
and survival. The P/C regimen markedly depleted both T and
B cells with relative sparing of myeloid cells. Readouts of
immunogenicity by the ICC assay showed 5/10 mice developed
antibodies in the IT only group, while no mice (0/10) developed
antibodies in either the P/C and R treated groups. However, the DC
assay revealed 3/10 mice treated with R indeed had anti-SS1P
antibodies present but 0/10 P/C-treated mice showed immunogenicity.
Although immune suppression by treatment with rapamycin
did cause reduced immunogenicity, antibodies to the anti-mesothelin
IT were detectable at low levels. On the other hand, immune
depletion with pentostatin plus cyclophosphamide prevented formation
of any detectable IT-targeted antibodies. These findings will
allow for the investigation of immune depletion with these drugs as a
mechanism to overcome immunogenicity in treatment of mesothelioma
with the immunotoxin SSIP. Since mesothelin is also
expressed on lung, ovarian and pancreatic cancer, results could be
applicable to treatment of these more common cancers as well.
Humanization of a High Affinity, Neutralizing Antibody
Against Hepatitis B Surface Antigen
Ashutosh Tiwari*, Navin Khannaw, Subrata Sinha*. *Biochemistry,
All India Institute of Medical Sciences; w Recombinant Gene Product
Lab, International Center for Genetic Engineering and Biotechnology,
New Delhi, India.
Hepatitis B virus chronically infect more than 500 million people
worldwide and account for about two-thirds of all hepatocellular
carcinoma, the third most common cause of cancer-related death.
Universal vaccination using Hepatitis B surface antigen (HBsAg) is
a possible way to achieve eradication of the disease. However,
alternative strategies to combat hepatitis B are needed because of
the existence of vaccine non-responders, emergence of viral escape
mutants and the persistence of a subpopulation of viral carriers in
developing countries. We previously generated a potentially
neutralizing mouse monoclonal antibody (5S) against HBsAg.
This antibody showed very high affinity and stable binding in
presence of different agents that usually destabilize antigenantibody
interactions. In this report we describe the humanization
of 5S antibody by grafting its antigen binding site onto framework
of the human consensus sequence of highest similarity. We have
used molecular modeling to alter not only the clearly permissible
residues but also several minimal positional template and VH/VL
interface residues. The humanized antibody retained a high binding
affinity (KD = 1.27 nmol/L) to HBsAg and bound to the same
epitope of HBsAg as the parent molecule. This high affinity
humanized antibody provides a basis for the development of
therapeutic molecules that can be safely utilized for the prophylaxis
and treatment for Hepatitis B infection.
Development of Cancer Stem Cell Vaccine
Toshihiko Torigoe, Yoshihiko Hirohashi, Akari Takahashi,
Satoshi Nishizawa, Rena Morita, Satoko Inoda, Noriyuki Sato.
Department of Pathology, Sapporo Medical University, Sapporo,
Japan.
Cancer stem cells (CSCs), a small population of cancer cells with
stem cell-like phenotype and tumor-initiating capacity, are
important targets for cancer therapy because they may be
responsible for recurrence after chemotherapy. We are exploring
the possibility for development of cancer vaccine targeting CSCs.
In order to clarify immunopathological properties of CSCs, CSCs
with high tumor-initiating capacity were collected from various
cancer cell lines by sorting a side population (SP), which has a
capacity to pump out Hoechst 33342 dye through ABC transporters.
By comparative gene expression analysis of SP and main
population (MP) cells derived from lung, breast and colon cancer
cell lines, several CSC genes were identified, including SOX2,
SMCP, DNAJB8 and OR7C1. These genes were expressed preferentially
in SP cells, but barely detected in normal adult tissues
except for testis. Overexpression of the genes increased tumorinitiating
capacity of cancer cells in vivo, whereas RNAi-mediated
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