Abstract Book 2010 - CIMT Annual Meeting

L125 Huijbers | New targets & new leads
Vaccination against the extra domain-B of fibronectin as a
novel tumor therapy
Elisabeth J. M. Huijbers 1 , Maria Ringvall 1 , Julia Femel 1 , Sebastian Kalamajski 1 , Agneta
Lukinius 2 , Magnus Åbrink 1 , Lars Hellman 3 and Anna-Karin Olsson 1
1 Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, SE-751 23 Uppsala, Sweden
2 Department of Medical Cell Biology, Uppsala University, BMC, SE-751 23 Uppsala, Sweden
3 Department Cell and Molecular Biology, Uppsala University, BMC, SE-751 24 Uppsala, Sweden
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During physiological angiogenesis (e.g. wound
healing and proliferation of the endometrium),
embryonic development and tumor angiogenesis
certain extra domains of the extracellular matrix
molecules fibronectin and tenascin-C are inserted
into the parent molecule by alternative splicing
leading to the formation of different isoforms.
Splice variants of both fibronectin and tenascin-C
are often co-expressed specifically around neovasculature
and tumor vasculature but have low or no
expression in normal adult tissue, rendering them
specific targets for anti-tumor therapy.
In our approach we have targeted the extra domain-
B (ED-B), which is a 91 amino acid domain alternatively
spliced into fibronectin and highly conserved
between species. It is identical in mouse, human,
rabbit, dog, monkey and several other species. To
evoke an immune response against the self-antigen
ED-B we used a recombinant fusion protein consisting
of a bacterial thioredoxin (TRX) part fused
with the extra domain B (ED-B), termed TRX-EDB.
This fusion protein has been injected together
with Freund’s adjuvant, a strong immunostimulator,
into eight-weeks old female wild type C57bl6
mice. Mice were boostered twice in a period of five
weeks before they were inoculated subcutaneously
with T241 fibrosarcoma cells, a tumor type known
to express ED-B. After a tumor growth period of
three weeks animals were sacrificed and blood and
tumors were removed. Nineteen out of 20 vaccinated
mice responded with production of anti-ED-B
antibodies and showed a 70% reduction in tumor
size compared to control animals injected with
Freund’s adjuvant and vehicle, lacking anti-ED-
B antibodies. Staining of murine grade III glioma
tissue, to see whether the serum from TRX-EDB
mice could detect native ED-B, showed an extensive
vascular staining pattern compared to normal brain
tissue, which is devoid of ED-B. This shows that
the anti-ED-B antibodies are able to detect native
tissue ED-B. Quantification of tumor necrotic area
revealed a tendency towards a greater necrotic area
in TRX-EDB injected compared to control animals.
Further investigation of the tumors with electron
microscopy revealed morphological changes of the
tumor vasculature of animals with anti-ED-B antibodies,
which was consistent with an immune
response towards the tumor vasculature expressing
ED-B. Moreover in tumors from animals with
anti-ED-B antibodies an increased number of infiltrating
neutrophils was observed, compared to
controls, confirming an immune attack of the vasculature.
Furthermore we detected an increased
amount of extravasated fibrinogen, indicative of
vascular leakage, in tumors of animals with anti-
ED-B antibodies compared to controls.
Therefore ED-B and possibly other tumor vascular
antigens such as the extra domain A of fibronectin
or the extra domain C of tenascin-C alone or in
combination are interesting candidate targets for
treatment of solid tumors. Since the choice of adjuvant
is of major importance for treatment success
in humans, we addressed this issue in a second
study and found a non-toxic biodegradable adjuvant
as potent as Freund’s in inducing an immune
response against a self-antigen (see poster by Julia
Femel et al.)