TARGETED AND UNTARGETED FUSION PROTEINS: CURRENT APPROACHES TO CANCER IMMUNOTHERAPY - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 296

TABLE 19.2 Combination Immunotherapy of TNT-3/IL-2
(20 ug) in RENCA-Bearing BALB/C Mice at 17 Days
Post-Tumor Implantation

fusion proteins produced only minimal improvement but the
combination containing the chTNT-3/IL-2 did show flat-
tening of the growth curve by day 17. Importantly,
LEC/chTNT-3 immunotherapy was found to be entirely
nontoxic (even at 100 m g), in marked contrast to the use
of chTNT-3/IL-2, chTNT-3/TNF- a , and TNT-3/mGM-CSF
fusion proteins, which demonstrated varying degrees of
toxicity when increased above 20 m g per dose. Treatment
with these three fusion proteins at doses exceeding 20 m g,
made the mice lethargic, induced a ruffled fur appearance,
produced a loss of appetite, and lowered activity levels of
treated mice.

Antibody Fusion Protein Dose (ug) Tumor Reduction (%)

Antibody alone control 20 0

chTNT-3/IL2 20 40

chTNT-3/muIFN- g 20 50

chTNT-3/TNF- a 20 25

chTNT-3/muGM-CSF 5 40

chTNT-3/muIFN- gþ
chTNT-3/TNF- a

20 þ 20 80

chTNT-3/muIFN- gþ
chTNT-3/muGM-CSF

20 þ 5 65

n-treated controls.

19.4.3 Co-Stimulatory Fusion Protein Combinations

Effective against multiple solid tumor types, TNFSF ligand
fusion proteins serve to provide second signal activation of
T cells and work well with T reg depletion. The co-stimulatory
abilities of these ligands may prove to be a powerful combi-
nation with, for example, the chemoattraction of the LEC
or the co-stimulation of the B7.1 and may possess an
unrivalled ability to attract and activate the immune cells
necessary for tumor regression and immunologic memory. As
described above, our laboratory had established the effective-
ness of B7.1-Fc N-terminal fusion protein in vivo in order to
determine whether it could be used in combination with
Fc-GITRL. As shown in Figure 19.8A, B7.1-Fc, in combina-
tion with Fc-GITRL, did indeed produce

models, this combination or the one in which muTNT-
3/muGM-CSF was substituted for the chTNT-3/IL-2, was
most effective. Since the tumors were found to continue
growing at about the same pace as control treated mice,
the therapeutic effect of these treatments was found to
be transient in nature. This incomplete immune response
may have been due to the nongeneration of immune
memory cells.

19.4.2 Chemokine and Cytokine Synergy

40-50% reduction
in tumor volume (COLON-26) in comparison to using
B7.1-Fc or Fc-GITRL alone.
In order to assess whether signaling through GITR and
CD137 receptors would lead to a synergistic antitumor
effect, a combination treatment was performed using Fc-
GITRL and Fc-CD137L. The data shown in Figure 19.8B
demonstrate that in comparison to their use alone, the
combination of these two C-terminal fusion proteins led
to

In order to enhance the therapeutic effectiveness of the
LEC/chTNT-3, Khawli et al. [91] focused their attention
on combining this reagent with other fusion proteins that
others have found to be critical components of cancer
immunotherapy. In this new combination therapy, groups
of MAD109-bearing BALB/c mice were injected with 20 m g
of LEC/chTNT-3 alone or with comparable doses of chTNT-
3/IFN- g , chTNT-3/TNF- a , chTNT-3/GM-CSF, or chTNT-
3/IL-2. After tumors reached 0.5 cm in diameter, all groups
were treated daily for 5 days and tumor growth was moni-
tored every other day by caliper measurement in three
dimensions. As shown in Table 19.3, combination therapy
with LEC/chTNT-3 and each of the four chTNT-3/cytokine

44-56% reduction in tumor volume (COLON 26) using
very low doses of each. In general, these data provide
evidence in mice that combination fusion proteins consisting
of these immune activators may indeed be especially effec-
tive for the immunotherapy of cancer.

TABLE 19.3 Combination Immunotherapy of LEC/chTNT-3
(20 ug) in MAD109-Bearing BALB/c Mice at 17 Days
Post-Tumor Implantation

19.5 MECHANISM OF ACTION:
IMMUNOREGULATORY T-CELL (T REG )
DEPLETION AND FUSION PROTEIN
COMBINATION THERAPY

Antibody Fusion Protein Dose (ug) Tumor Reduction (%)

Antibody alone control 20 0

T-cell anergy is one of the major impediments to the ability
of the immune system to eradicate established tumors. To
elicit an effective T-cell response, the engagement of the
T-cell receptor by peptide-major histocompatibility complex
needs to occur in the setting of a second signal provided by
co-stimulation.

LEC/chTNT-3 20 40

chTNT-3/muGM-CSF 20 55

chTNT-3/TNF- a 20 55

chTNT-3/muIFN- g 20 55

chTNT-3/IL-2 20 70

In order to create more effective