| Abstract |
Background: GDF-15 is a divergent member of the TGF-superfamily, which was
first described as macrophage inhibitory cytokine-1 (MIC-1), revealing an
immune modulatory function. GDF-15 is a soluble protein which is, under
physiological conditions, highly expressed in the placenta and found in
elevated levels in blood sera of pregnant women. Apart from the placenta,
GDF-15 is expressed in healthy tissue, albeit to a lower extent and
overexpressed in many solid tumors. A variety of different functions are
attributed to GDF-15 in healthy as well as diseased humans. On the one
hand, GDF-15 is required for successful pregnancy and low GDF-15 serum
levels during pregnancy correlate with fetal abortion. On the other hand,
overexpression of GDF-15, which can be observed in several malignancies is
correlated with a poor prognosis. Furthermore, tumor derived GDF-15 leads
to cancer associated anorexia-cachexia syndrome in mice. The aim of my PhD
thesis was to further investigate the role of GDF-15 as an immune
modulatory factor in cancer, in particular, by inhibiting the target
molecule in vitro and in vivo. Therefore, the main focus was placed on the
generation and characterization of monoclonal GDF-15 specific blocking
antibodies, which were tested in vitro and in vivo, which represents a
substantial part of my work.
Results: Here, GDF-15 was shown to be highly expressed in human
gynecological cancer and brain tumors. We could then demonstrate that GDF-15
modulates effector immune cells in vitro. GDF-15 mediated a slight
downregulation of the activating NKG2D receptor on NK and CD8+ T cells,
which is crucial for proper anti-tumoral immune responses. Furthermore, we
could demonstrate that GDF-15 reduces the adhesion of CD4+ and CD8+ T
cells on endothelial cells in vitro. A negatively affected trans-
endothelial migration of leukocytes into inflamed tissue could explain the
low T cell infiltration in GDF-15 expressing tumors, which were observed
in vivo, where mice bearing (shRNA mediated) GDF-15 deficient glioma cells
revealed enhanced immune cell infiltrates in the tumor microenvironment,
compared with the GDF-15 expressing control group. Those animals further
exhibited a decreased tumor growth and prolonged survival. GDF-15 is a
soluble protein, secreted by more than 50% of solid tumors and associated
with grade of malignancy. Therefore a neutralizing monoclonal antibody to
GDF-15 was assumed to be an auspicious therapeutically anti-cancer tool.
Search an antibody was thus generated in GDF-15 knock out mice against
human GFD-15. Amongst many clones, the GDF-15 antibody clone B1-23 was
found to be applicable in Western blot as well as in ELISA techniques,
detecting a three-dimensional epitope of the mature GDF-15 dimer with high
affinity and specificity. To enable the humanization for a later
administration in humans, the variable regions of antibody B1-23 were
identified by a special PCR method using degenerate primers and cloned
into a sequencing vector. The sequence obtained thereby enabled the
generation of chimeric and humanized B1-23 variants. After further
comprehensive characterization, the original mouse antibody B1-23 as well
as the chimeric antibody (ChimB1-23) and the humanized B1-23 antibody
(H1L5) were applied in a melanoma xenograft study in vivo. None of the
antibodies could significantly inhibit tumor growth. However of utmost
importance, body weight loss mediated by tumor derived GDF-15 could be
significantly prevented upon administration of all three GDF-15 specific
antibodies, which confirmed the antagonizing functionality of the
immunoglobulin.
Conclusions: GDF-15 is a promising cancer target, involved in tumor
progression and cancer related cachexia. A monoclonal GDF-15 antibody was
generated, which served on one hand as a tool for molecular biological
applications (Western Blot, ELISA, etc.) and on the other hand was applied
as an antagonizing antibody in vitro and in vivo. Even though tumor growth
inhibition by GDF-15 depletion in T cell deficient athymic mice failed
using B1-23, the same antibody and derivatives thereof (chimeric and
humanized) impressively prevented tumor associated cachexia in UACC-257
melanoma bearing nude mice. The missing anti-tumor effect in our own
melanoma model in nude mice can only partially be explained by the missing
secondary immunity, in particular cytotoxic T cells, in the athymic
animals, since in a similar melanoma model, performed by an external
company, a tumor reduction in immunocompromised animals was observed when
B1-23 was administered. These findings support the idea that T cells are
substantial for an effective tumor immunity and are in line with the
results of the syngeneic, T cell comprising, mouse glioma model, where
silencing of tumor expressed GDF-15 led to an enhanced intratumoral T cell
infiltration and a prolonged survival. Taken together our data allow for
the conclusion that tumor associated cachexia can be combatted with the
GDF-15 antibody B1-23. Further, B1-23 might elicit direct anti-tumor
effects in immune competent models, which contain T cells, rather than in
an athymic, T cell deficient nude mouse model.
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