I think the complete discussion section of this paper is worth reading.
4. Discussion
We demonstrate that 2aG4-IL2 treatment of irradiated 4T1
tumor cells significantly increases their ability to function as a vaccine.
The enhancement in immunogenicity is attributable to three
mechanisms. First, the improvement is antigen-specific indicating
that 2aG4-IL2 blocks PS-dependent immunosuppressive signals.
Second, the improvement is greater with 2aG4-IL2 than with 2aG4
alone, indicating that immunostimulatory cytokine-IL2 contributes
to the vaccination. IL-2 is a potent T-cell growth factor that has been
demonstrated in several prior studies to function as a vaccine adjuvant
[30–32]. Third, although not formally demonstrated here, we
expect that the Fc portion of 2aG4-IL2 enhances presentation of
tumor antigens by stimulating Fc receptor mediated phagocytosis
by antigen presenting cells, as demonstrated previously in other
systems [33,34].
A few PS blocking strategies to restore the immunogenicity of
tumor cell vaccines have been reported. Masking PS on cancer cell
vaccines with the PS-binding protein annexin 5A has been reported
to reverse PS mediated immunosuppression and improve antitumor
immune responses [15,16]. Injection of annexin 5A coated
tumor cells in combination with radiotherapy resulted in a strong
anti-tumor effect, with 90% of the mice rejecting the tumor after
challenge as opposed to only 25% of control animals. Similarly,
blockade of PS-dependent phagocytosis through the MFG-E8 pathway
significantly increased the antitumor immunity induced by
a GM-CSF-secreting tumor cell vaccine [35]. However, given that
multiple receptors exist for PS on APC [36–39], interfering with
MFG-E8 pathway alone may not be sufficient to block PS mediated
suppression completely. The anti-PS antibody strategy for
masking PS has an advantage over the annexin 5A and MFG-E8
strategies in that it promotes efficient antigen uptake and processing
through Fc receptor-mediated phagocytosis, which favors the
cross-priming pathway that preferentially activates CD8+ cytotoxic
T cells responses in vivo [40,41].
We recently reported that treatment of rats bearing established
F98 gliomas in their brains with unconjugated 2aG4 and
local irradiation cured 15% of the animals. Surviving animals were
immune to rechallenge with viable F98 tumor cells[10]. In vitro,
2aG4 enhanced the ability of dendritic cells to generate F98-specific
cytotoxic T-cells in an antigen-specific manner. Consistent with
these findings, our current study shows that masking PS on irradiated
4T1 cells with unconjugated 2aG4 enhanced antitumor CTL
immunity, and reduced spontaneous metastasis, although, no survival
benefit was observed. However, when the 2aG4 was fused
to IL-2 and used to coat 4T1, a powerful immune response to 4T1
tumor cells was induced. Immunostimulatory cytokines, such as
IL-2, have been extensively used to boost the antitumor immune
response of cancer vaccines in other studies [42–45].
IL-2 stimulates the expansion of antigen-activated T-cells, but
also stimulates the expansion of regulatory T-cells (Tregs) which
can be detrimental to a vaccine. It has been reported that brief
low-dose IL-2 treatment favors expansion of Treg [46]. The balance
of effects on helper T-cells and NK cells versus Tregs is probably
determined by the concentration of IL-2, timing and duration of
exposure. Indeed, superior antitumor effects with cancer cell vaccines
have been observed when IL-2 adjuvant is formulated as a
slowly IL-2-releasing cytokine depot rather than as free IL-2 [47].
Our 2aG4-IL-2 fusion has a longer in vivo half life than does free IL-
2 and would therefore persist at higher concentrations for longer,
which may explain its beneficial immune activating properties. An
alternative strategy to avoid the detrimental effects of IL-2 on Tregs
would be to fuse 2aG4 to a cytokine that is free from this property,
such as IL-15 or IL-21.
In summary, we have shown that a whole cell vaccine created
by coating irradiated tumor cells with anti-PS-IL2 fusion protein
can generate robust anti-tumor immunity. While this study was in
a prophylactic setting, future studies will address the therapeutic
efficacy of this approach in animals from which the primary tumor
has been removed surgically. This treatment setting could begin to
address whether patients who have residual diseases and who are
at high-risk to develop cancer might benefit from immunization
with their own killed tumor cells coated with anti-PS-IL2 [48,49].
Given the broad action of PS-dependent immunosuppression, and
the fact that all killed whole cell vaccines strongly express PS, the
current approach could be generally applicable. It might also be
applicable in a so-called in situ vaccine setting in which a patient’s
tumor is treated with irradiation or other local therapy and anti-
PS-IL2 then injected into the dead tumor tissue [50,51]. Since most
chemotherapeutic drugs and radiation elicit apoptotic cell death, PS
exposed on dead and dying tumor cells would be expected to abrogate
local immunity; if so, intra-tumoral injection of anti-PS-IL2
might simultaneously overcome the immunosuppressive environment
while adding IL-2 to activate immunity
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