TUMOR-TARGETED SUPERANTIGENS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 366

TABLE 24.3 Therapy of Tumor Metastases in the B16-EpCAM Tumor Model with C215Fab-SEA in Combination with
Gemcitabine

Mean Number of Lung Metastases
Day 28 (n=8)

Mean Number of Lung Metastases
Day 40 (n=8) Treatment

Control a 117 All animals dead

C215-SEA (10 m g/mouse i.v. days 3-6) 23 21

Gemcitabine (2.4 mg/mouse i.p. days 8, 11, 14, 17) 141 All animals dead

11 b 10 c C215Fab-SEA þ gemcitabine

a Nontreated tumor inoculated animals.
b p < 0.001, comparison to gemcitabine alone.
c p < 0.05, comparison to C215FabSEA alone.

shown [62,72,73] and could potentially have played a posi-
tive role inducing TTS effect enhancement. As was shown in
the clinical COMBO study with naptumomab estafenatox
and in preclinical models, docetaxel reduced production of
potentially neutralizing anti-SAg antibodies when given in
certain timely combinations with TTS [22].
Preclinical results indicate that T lymphocyte reactiva-
tion with TTS may be enhanced by interference with
immune suppression and anergy. In attempts to enhance
T lymphocyte antitumor effects, we have investigated com-
binations of TTS with IL-2 [28], IFN- a [21], and the mAbs
recognizing CD25, CTLA-4, and Gr-1 [74]. These immune
modulators have been shown to enhance immune function or
inhibit immune suppression, and in timely combinations
with C215Fab-SEA in the B16-EpCAM tumor model, they
all show synergistic antitumor activity (Figure 24.7). IL-2
and IFN- a are approved for the treatment of multiple
cancers and exerts immunomodulatory effects, which
make them appropriate therapeutics to combine with TTS
therapy. IL-2 and IFN- a enhanced and sustained CD8

myeloid-derived suppressor cells (MDSCs). Treatment with
depleting anti-CD25 or anti-Gr-1 mAbs before the C215Fab-
SEA cycle resulted in synergistic antitumor effects similar to
the effects recordedwith the blockinganti-CTLA-4mAbwhen
given during the C215Fab-SEA cycle (Figure 24.7). Within
48 h after C215Fab-SEA treatment, EpCAM expressing
tumors were infiltrated and targeted by CD4 þ and CD8 þ T
cells. Inparallel, CD3 þ CD4 þ Foxp3 þ T reg cellswere expanded
and infiltrated the tumors as potential modulators of the
powerful SAg induced Th1 response. The TTS induced anti-
tumor activity was dampened and short-term reactivation was
inhibited possibly due to the suppressive effects by the T reg
cells. The expansion of T reg cells was inhibited by anti-CTLA-
4 treatment, and the CTL activity was enhanced and easily
reactivated by a second TTS cycle. The combination of
C215Fab-SEA with anti-CTLA-4 for treatment of B16-
EpCAM tumors growing in the lungs of syngeneic mice
showed classic synergy as compared to the monotherapies
(Figure 24.7). CD11b þ Gr-1 þ Ly6C int Ly6G high and
CD11b þ Gr-1 þ Ly6C high Ly6G MDSCs increased in numbers
in the spleen and in thevicinityof lung tumors in tumor bearing
mice at 72-96 h after initiating TTS treatment. To interfere
with these potentially suppressive cells, we used a depleting
anti-Gr-1 mAb. The combination of C215Fab-SEAwith anti-
Gr-1 for treatment of B16-EpCAM tumors growing in the
lungs of C57Bl/6 mice showed synergy as compared to the
monotherapies. The antitumor synergywas seenwhen the anti-
Gr-1 mAb was given the day before start of TTS treatment but
not when given the day after last TTS injection. Furthermore,
when an anti-Ly6G mAb was given in a similar setting, no
additive effects were recorded indicating that the CD11b þ Gr-
1 þ Ly6C high Ly6G monocyticMDSCs represented the impor-
tant cell type to target for therapy enhancement. Therefore,
antitumor effects by TTS therapy can be greatly enhanced by
interfering with T reg cells and MDSC in tumor models sug-
gesting similar beneficial effects in human clinical settings.
Furthermore and to evaluate naptumomab estafenatox in
models for combination treatments with anti-angiogenic and
cytostaic drugs, SCID mice were used. The compatibility of,
and additive antitumor effects by the combination of naptu-
momab estafenatox with bevacizumab, docetaxel, and

T-
cell activation induced by treatment with the TTS C215Fab-
SEA in the B16-EpCAM model, as reflected by increased
and prolonged CTL activity as well as by augmented serum
IFN- g levels. C215Fab-SEA synergized with IL-2 and IFN-
a in inhibiting tumor growth of pulmonary metastases of
EpCAM-expressing B16 mouse melanoma cells as com-
pared to monotherapy. In a long-term survival experiment
using the same tumor model, the prolonged median survival
time was dramatically increased with the combination of
C215Fab-SEA and IFN- a as compared to monotherapy
(Figure 24.7). Hence, the combination treatment provoked
synergistic antitumor effects as measured by the number of
lung tumors and markedly prolonged survival and the
enhanced therapeutic efficacy correlated with a striking
and sustained increase of CD8- and perforin-expressing
tumor-infiltrating cells.
MAbs recognizing CD25, CTLA-4, and Gr-1 were used
alone and in timely distinct combinations with C215Fab-SEA
in the B16-EpCAM tumor model. CD25 and CTLA-4 are
expressed by regulatoryT (T reg ) cells (alsoexpressingCD4and
Foxp3), and Gr-1 is expressed by granulocytic and monocytic

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