TUMOR-TARGETED SUPERANTIGENS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 359

was associated with a most potent T-cell activation, and
therefore, it was necessary to give doses as low as a few
nanogram per kilogram to avoid unacceptable toxicity. In
addition, the dose had to be individualized based on
preformed anti-SAg (anti-SEA) antibodies. Primum movens
for TTS function is TCR binding and T-cell activation. Wild-
type SAgs bind to MHC class II cell membrane proteins, and
the complexes subsequently bind to TCRs containing certain
V b sequences and activate these T cells. After proliferation
and differentiation, the T lymphocytes are capable to infil-
trate the tumor tissue presenting the TTS fusion proteins
specifically bound to the tumor cells. These T cells are
activated in the tumor due to TCR-binding to the SAgs of the
TTS protein, produce cytokines, and perform direct per-
forin-dependent tumor killing. To achieve maximal targeted
antitumor effects, a balanced relationship between binding
affinities of the three functional binding sites in the TTS is
required. The objective to mimic the T-effector cell (e.g., the
CTL) interaction to a tumor target cell, the TCR binding to
the MHC/tumor peptide target, drove the development to the
TTS naptumomab estafenatox showing distinct character-
istics regarding binding to the tumor antigen (5T4), the TCR,
and to the MHC class II proteins [18] (Hedlund et al., to be
published). The optimal TTS should have very high affinity
for the tumor antigen, intermediate/low affinity for TCR,
and very low affinity for MHC class II products.
A fusion protein consisting of the 5T4Fab moiety fused to
SEAwith a point mutation decreasing MHC class II protein
binding (associated to decreased toxicity), anatumomab
mafenatox [24], has been investigated in clinical Phase I
and Phase II trials with very encouraging results [38,58] (see
24.5.1). However, this fusion protein was also associated
with some of the drawbacks seen with nacolomab tafenatox,
necessity to give low doses as well as individualized dosing
on the basis of preformed anti-SAg (anti-SEA) antibodies.
Affinity of distinct SAgs to MHC class II proteins is
correlated to T-cell activation capacity, toxicity, and capacity
to direct CTLs in SAg-dependent cell-mediated cytotoxicity
(SDCC, killing of MHC class II protein expressing target
cells incubated with SAg). The targeting of CTLs by a TTS
fusion protein to tumor cells can be measured in assays
for SAg-antibody-dependent cell-mediated cytotoxicity
(SADCC; killing of target cells incubated with TTS fusion
protein specifically bound to target with the mAb part). TTS
fusion proteins stimulate human CTLs to kill tumor cells
in a dose-dependent manner. Naptumomab estafenatox dis-
played an increased affinity (by more than a factor 1.5) to the
5T4 antigen as compared to the preceding compound ana-
tumomab mafenatox [18]. The affintiy for naptumomab
estafenatox was in the order of 1.6 nM. Naptumomab esta-
fenatox was

naptumomab estafenatox displayed 100-fold and 10,000-
fold decreased activity when compared with anatumomab
mafenatox and 5T4Fab-SEA (SAg similar to nacolomab
tafenatox), respectively, owing to decreased MHC class II
binding. Naptumomab estafenatox induced an MHC class II
and dose dependent proliferation in human lymphocytes,
however, 50-fold and

1000-fold higher concentrations
were needed compared to anatumomab mafenatox and to
a fusion protein with wild-type SEA, respectively [18].
Naptumomab estafenatox mediated a 1000-fold more potent
5T4 targeted cytotoxicity (EC 50

>

10 11 M) compared with
the MHC class II directed cytotoxicity (EC 50 10 8 M) [18]

(Figure 24.3). Furthermore, preformed human anti-SAg anti-
bodies do not recognize the functional regions in the SAg part
of naptumomab estafenatox to the same extent as in the
predecessor construct [18]. A 10-fold higher concentration of
anti-SAg (anti-SEA) antibodies was needed to neutralize the
bioactivity of naptumomab estafenatox compared with ana-
tumomab mafenatox measured as inhibition of SADCC
activity. These findings show that the activity of naptumomab
estafenatox is significantly less inhibited by human anti-SAg
than that of anatumomab mafenatox. Using various in vitro
assays, it is estimated that only 10-15% of human anti-SEA
antibodies can bind naptumomab estafenatox [19].
Thus, even though the novel TTS naptumomab estafe-
natox has most potent antitumor properties, it contains a
SAg moiety without classic SAg activity. The pivotal res-
cued functional part of the two hybridized and mutated
Staphylococcal enterotoxins (A and E) is the TCR-binding
region. The MHC class II binding capacity is minimal and
the T-cell proliferation induced by naptumomab estafenatox
has an EC 50 > 1000 times higher than conventional wild-

type SAgs resulting in dramatically reduced toxicity.
Furthermore, the target epitopes for naturally occurring
anti-SAg antibodies were reduced and dosing of naptumo-
mab estafenatox was therefore considered to be independent
of baseline anti-SAg antibodies.

24.5 CLINICAL EXPERIENCE WITH TTS
THERAPEUTIC FUSION PROTEINS

Three TTS therapeutic fusion proteins have been investigated
in the clinic: nacolomab tafenatox, anatumomab mafenatox,
and naptumomab estafenatox. The latter two contain Fab
moieties recognizing the 5T4 antigen and engineered SAgs.
The 5T4 antigen is expressed by essentially all NSCLC, PC,
and RCC tumors (Table 24.2), and the clinical trials have so
far been focused on patients having these tumor types.

50% more efficient as compared with anatu-
momab mafenatox, in killing human 5T4-positive Caki-2
tumor cells as measured by SADCC (Figure 24.3). In the
killing of MHC class II positive Raji cells by SDCC,

24.5.1 Anatumomab Mafenatox

Two Phase I studies (USA study andEurope study) to evaluate
anatumomab mafenatox in patients with advanced NSCLC