Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

186 9 Dendritic Cells and Cancer: Prospects for Cancer Vaccination
Blood DCs may be obtained as part of a mononuclear cell preparation and then isolated
via a range of techniques. Gradient techniques have been developed commercially
[107]. Given the potential for other cell populations to contribute confounding
regulatory influences, a relatively pure DC population seems advisable in the longer
term. Attempts to use mAb-based selection of DC have therefore been initiated in at
least two or three laboratories. Using the CMRF-44 [108] or CMRF-56 mAb it appears
that reasonable numbers of relatively pure DCs can be obtained (Lopez et al.,
submitted). Of course, this method of selection allows for DC subset selection, and
this might allow both stimulatory and regulatory DC preparations to be obtained.
Despite the similarities, there are notable differences between Mo-DCs and myeloid
CD11c + DCs. Differences include expression of presumed regulatory Ig family molecules
(Clark et al., in preparation) and cell surface lectins, e. g. CD205, CD206 and
CD209 [20]. Functional data [109] suggests that myeloid blood CD11c + are capable of
inducing a greater percentage of effector T lymphocytes in vitro and that these DC
preparations should be assessed independently.
Whilst there are established methods for purifying stem cells using CD34 or CD133
reagents for antibody-based cell selection, this, plus the mobilization, are significant
preliminary steps before the in vitro culture and generation of DCs. One of the attractive
features of Mo-DCs to investigators is that venesection may provide enough cells
[101]; however, there is an increasing trend to use apheresis procedures to obtain larger
number of mononuclear cells for the in vitro cultures [76]. Apheresis will certainly
be essential if blood DCs are to be used but recent data suggests that they ªmobilizeº
during the procedure [110] and that sufficient numbers will be available even
without mobilization in cancer patients (Vuckovic et al., submitted). Quality control
of these preparations will become a big issue as raised elsewhere [111].
9.6
Loading DC with Antigens
Having chosen the DC preparation for immunotherapy, the next challenge is to optimize
the conditions for antigen loading. The state of Mo-DC maturation, the media,
and the presence and type of serum/protein additives all make a difference [76, 101].
However, perhaps one of the more important variables is the time of incubation ±
this not only influences antigen loading, but may also determine the type of immune
responses initiated. These variables are also important for blood DC preparations
[104].
The choice of methodology for providing the antigen is critical. The field began with
irradiated tumor cells or tumor cell lysates or partial peptide preparations. These
were appropriate to initiate investigations, but they are difficult to standardize and
may contain critical DC-activating or -modulating capacity, e.g. heat shock proteins,
that needs better defining. Now the cell biology revolution has given us the opportunity
to consider many different potential forms of TAAs.
TAA peptides, which bind class I and class II, are now readily predicted and validated.
They are easy to manufacture and produce under GMP conditions. They can