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Cellular Immunotherapy


Study of the relationship between augmentation of NKT cells and
induction of adaptive immunity using a new ligand, alfa-C-GalCer

alfa-GalCer is a prototype compound used for studies of the presentation of glycolipids
on CD1d molecules NKT lymphocytes. A single intravenous dose of glycolipid triggers
the production of several cytokines over the course of one day, the short-lived
activation of NKT and NK cells and a prolonged adaptive T cell immune response if
certain antigens are given together with alfa-GalCer. Recently, CD1d-binding
glycolipid analogs including sphingolipids and sulfative variants have been synthesized.
These molecules induce the response of NKT cells with the secretion of IFN-gamma and
IL-4 in human and mouse. One of the CD1d-binding glycolipid analogs, alfa-GalCer, is
known to be much more active than alfa-GalCer in inducing resistance to coadministered
tumor cells and malaria parasites. We determined if the more active glycolipid alfa-C-GalCer
is a more potent inducer of DC function in various forms of
innate and adaptive resistance.

We found that a new analogue,alfa-GalCer, potently induces these innate and adaptive
immune responses in mice. alfa-GalCer triggers IL-12 and IFN-gamma production more
effectively than alfa-GalCer, whereas it minimally elicits IL-4 and TNF-alfa release
into the serum. Alfa-C-GalCer also better mobilizes NKT and NK cells to resist B16
melanoma than alfa-GalCer. In addition, we found that DCs loaded with alfa-C-GalCer
induce NKT cell response in vivo with greater magnitude and longer kinetics than DCs
loaded with alfa-GalCer. Such an effect could be attributed to the stable binding of
alfa-C-GalCer to DCs compared with alfa-GalCer. When glycolipid is targeted to DCs
in spleen together with antigens in dying cells, such as irradiated tumor cells, alfa-
C-GalCer is active as an adjuvant for T-cell immunity at as low dose as 20ng/mouse,
where it is also able to upregulate the required CD40L costimulatory molecule on NKT
cells. Therefore, alfa-C-GalCer a glycolipid that binds more stably to DCs and acts as
a more effective link between represents innate and adaptive immunities in vivo.

Evaluation of function of human invariant NKT cells from cancer patients
NKT cells play a role in the immunological regulation of certain disease, and their
frequency and/or function may be related to disease prognosis. However, it is
often difficult to evaluate NKT cell function in patients with malignancies due
to the reduced numbers of NKT cells as well as the dysfunction of APCs used as
stimulators. To detect NKT cell function independent of human APCs, we focused
on a homogenous population of murine DCs. NKT cells recognize glycolipids
presented by the nonpolymorphic MHC class I-like molecule CD1d, which is related
to beta2-microglobulin-associated transmembrane proteins. The high sequence
homology between human and murine CD1d (60.4% for the alfa1 domain and 62.4% for
the alfa2 domain) allows human V alfa24+ NKT cells to respond to murine CD1d. Our
goal is to find an approach to evaluate the function of human NKT cells by
comparing human and murine DCs.

We found that NKT cell function could not be evaluated by conventional ELISPOT
assays, confirming the impaired function of APCs in patients with chronic
myelogenous leukemia (CML) in the chronic phase. To overcome this problem, we
have established a sensitive assay using murine DCs to evaluate the function
of small numbers of human NKT (hNKT) cells in the absence of autologous APCs.
We found that CML patients in the chronic phase and under medication with
imatinib showed complete cytogenetic response (CCR) and had hNKT cells
appeared to be nonfunctional in patients partially responsive (PR) to imatinib
treatment, who did not produce IFN-γ upon stimulation. The results indicate
the possibility of developing effective immunotherapy through the generation
of functionally potent hNKT cells in ex vivo by alfa-GalCer-pulsed DCs.

Phase I and IIa clinical study with NKT Cell Therapy in Lung Cancer
In collaboration with Chiba University, clinical studies using alfa-GalCer-pulsed
autologous DC therapy have been carried out for the patients with advanced non-
small cell lung cancer. We found no remarkable adverse effects as a primary
endpoint in 20 patients totally in Phase I/IIa study. In our analyses at the
present stage, we have got encouraging immunological and clinical results in
phase I/IIa trials. This project is undertaken in collaboration with Drs.
Shinichiro Motohashi, Toshinori Nakayama, Takehiko Fujisawa (Chiba Univ.) and
Masaru Taniguchi (RIKEN).