SOLUBLE T-CELL ANTIGEN RECEPTORS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 462

cells by flow cytometry and confocal microscopy methods
[28]. Using this reagent, Pien et al. [28] demonstrated that
low levels of AAV capsid-derived antigen are endogenously
presented on human hepatocytes following AAV transduc-
tion. The STAR reagent could also be used to protect
transduced hepatocytes from CTL-mediated cytolysis
directed against cognate and cross-reactive epitopes derived
from different AAV serotypes. These results provide insight
as to how low levels of capsid antigen presentation and
subsequent immune responses may limit the effectiveness
of AAV-based gene therapies [28]. Additionally, STAR
Multimers have been used to quantitate antigen presentation
in addressing more fundamental questions on the relation-
ships between antigen dose and events leading to T-cell
activation [29]. Together with their potential in clinical
diagnostic and vaccine development applications described
earlier, these studies confirm that STAR reagents can be used
in simple, direct, sensitive, and quantitative methods to
advance the understanding of endogenous antigen presenta-
tion and related immune responses.

clinically relevant tumor models [17,23]. Nude mice bearing
primary tumors were injected intravenously (i.v.) with
264scTCR/IL-2 (from 0.15 to 1.6mg/kg), an equivalent
dose of IL-2, or PBS daily for 4 days, followed by treatment
every other day for a total of nine doses. With this treatment
regimen, 264scTCR/IL-2 inhibited the growth of sub-
cutaneous tumors derived from the A375 melanoma cells
in a dose-dependent manner and exhibited significantly
better antitumor activity than IL-2 alone. Moreover, treat-
ment with 264scTCR/IL-2 resulted in significant tumor
growth retardation in mice bearing large A375 tumors
(Figure 31.3A) [17]. The chimeric ALT-801 fusion protein
was also shown to exhibit potent dose-dependent antitumor
activity, equivalent to that of murine version, in this model
(Figure 31.3B) [17]. In addition, the p53-specific
264scTCR/IL-2 fusion was significantly more effective
than IL-2 in inhibiting the growth of tumors derived from
p53 þ /HLA-A 0201 þ PANC1 human pancreatic adeno-
carcinoma cells (Figure 31.3C) and p53 þ /HLA-A 0201 þ
MDA-MB-231 human mammary adenocarcinoma. How-
ever, tumor outgrowth is not inhibited in 264scTCR/IL-2
treated mice bearing HLA-A 0201-negative human
tumors (Figure 31.3D), confirming that antigen targeting
plays a role in the efficacy of 264scTCR/IL-2 against
p53 þ /HLA-A2 þ tumors [17]. It also demonstrates that
ALT-801 has substantial antitumor activity against p53 þ
tumors regardless of the tumor origin.
A major limitation with recombinant human IL-2 therapy
is the thrice-daily i.v. dosing regimen required due to the
molecule's short circulatory half-life. In contrast, evaluation
of ALT-801 in a humanized HLA-A2.1/K b -transgenic
mouse strain indicated that the fusion protein has a fivefold
longer terminal half-life (

31.4 PRECLINICAL TESTING USING STAR
FUSION PROTEINS

31.4.1 Preclinical Efficacy, Pharmacokinetic,
and Toxicity Studies

ALT-801 and its original nonchimeric murine version,
264scTCR/IL-2, have been extensively characterized in
vitro as described early [5,17]. Initial studies of
264scTCR/IL-2 showed that it retains the ability to specifi-
cally recognize the p53 (aa264-272) peptide displayed in
the context of HLA-A 0201 on peptide-loaded cells as well
as on melanoma cells and that the IL-2 portion of the
molecule exhibits full biological activity [5]. The fusion
protein also was capable of mediating conjugation of
effector and target cells bearing the appropriate IL-2 recep-
tor and p53 peptide/HLA-A 0201 complexes. The affinity
(K D ) of the 264scTCR/IL-2 protein for p53 peptide/HLA-
A 0201 complex was determined to be in the range of
10 6 -10 7 M [5]. Similar analysis carried out with
ALT-801 indicated that it retained both the TCR binding
activity and IL-2 biological activity of the 264scTCR/IL-2
fusion protein [17].
The antitumor activities of ALT-801 and 264scTCR/IL-2
fusion proteins have been evaluated in xenograft tumor
models in immunocompromised mice [5,17,23]. Initially,
we demonstrated that 264scTCR/IL-2 treatment could sig-
nificantly reduce tumor burden in an experimental mouse
model of lung metastasis with p53 þ /HLA-A 0201 þ A375
human melanoma cells [5]. Studies were also conducted to
directly compare the antitumor activity of the p53-specific
scTCR/IL-2 fusions to recombinant human IL-2 in more

2 h vs 0.4 h: IL-2) and is cleared

90-fold slower (0.5mL/h vs 42.0 mL/h: IL-2) than IL-2
[23]. Results of these studies also indicate that the scTCR
and IL-2 domains of ALT-801 are not cleaved in vivo, but
instead remain stably associated throughout the in vivo
metabolism and clearance of ALT-801. Thus, it is clear
that the generation of the fusion between the IL-2 and the
scTCR domain provides a significant improvement in
the pharmacokinetic profile of the cytokine, consistent
with the results observed with other cytokine fusion proteins
[17,23,40].
To understand differential effects of tumor antigen tar-
geting and enhanced pharmacokinetics on ALT-801 efficacy,
A375 tumor bearing mice were treated with ALT-801 or a
control scTCR/IL-2 fusion, MART1scTCR/IL-2, which
does not recognize A375 tumor cells. The results shown
in Figure 31.3E indicate that ALT-801 treatment continues
to exhibit potent antitumor activity compared to PBS and
that MART1scTCR/IL-2 provides intermediate but signifi-
cant inhibition of A375 tumor growth in this model. In
separate studies, MART1scTCR/IL-2 was found to display
equivalent IL-2 bioactivity as ALT-801 and a serum half-life

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