SOLUBLE T-CELL ANTIGEN RECEPTORS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 458

tumors, and IFN- a (Pegasys 1 ) and small molecule viral
inhibitors for chronic viral infections in humans. We are also
testing the bispecific fusions linking the scTCR domains to
anti-CD3 antibodies or IgG Fc effector domains as a means
of redirecting T cell and natural killer (NK) cell responses,
respectively, against tumors. As described in the following
section, the fused cytokine and antibody domains retain their
native functional activity and in many cases, exhibit
improved pharmacokinetics in vivo. Thus, the scTCR-based
fusions are being employed in a drug delivery system to
enhance efficacy and lower the therapeutic dosage of cur-
rently approved drugs and to provide more convenient
dosing regimens with decreased toxic side effects.

detectable labels. The increased avidity of STAR Multimers
results in an apparent higher pMHC binding affinity than
that observed for the monomeric scTCR [15]. Thus, STAR
Multimers are able to specifically and stably bind endoge-
nous pMHC complexes under various experimental condi-
tions [15]. We are exploring similar approaches using
multiple copies of the scTCR to coat the surface of nano-
particles. These targeted nanoparticles containing therapeu-
tic drugs have been found to be effective in reducing growth
of tumors bearing the TCR-specific pMHC antigen in animal
models.
Fusion of the scTCR domains to functional protein
scaffold domains, such as the IgG Fc effector domains
described earlier, have been used to create dimeric anti-
body-like molecules [14]. We have expanded this strategy to
novel functional scaffolds including soluble dimerization
domains of the IL-15/IL-15 receptor a (IL-15R a ) complex
[22]. In each case, these multimeric scTCR fusions exhibit
higher functional affinity to their cognate pMHC molecules
than the monomeric form and can effectively target effector
activity of the scaffold domains against cells expressing the
pMHC. Additionally, we have found that scTCR fusions to
other receptor binding proteins (i.e., cytokines or scAbs) can
be used in vitro or in vivo to decorate receptor-positive cells
with multiple scTCR binding domains. These cells are then
able to specifically recognize the pMHC-positive target cells
at the disease site in much the same manner as a native TCR-
bearing T cell [23]. These findings, described in more detail
in the following section, indicate that high-affinity inter-
actions between monomeric TCR and pMHC proteins are
not required for effective localization and retention of the
dimeric/multimeric scTCRs or the scTCR-coated immune
cells in target tissues carrying the cognate pMHC com-
plexes [23]. Such results are consistent with recent
theoretical models and experimental data of tumor uptake
obtained using targeting molecules in different sizes and
affinities [24].

31.1.2 Mutagenesis and Multimerization for Improved
Target Recognition

Naturally occurring TCRs generally interact with pMHC
complexes at relatively low affinities, typically in the range
of 1-100 m M [1]. This is due to the lack of somatic hyper-
mutation and affinity maturation during T-cell development,
the natural process of positive and negative selection of
T-cells in the thymus, and the role of other membrane-bound
co-receptors in stabilizing and augmenting TCR-mediated
signaling. Several mutagenesis and selection approaches
including phage, yeast, and mammalian cell display tech-
nologies have been employed to increase the affinity of the
TCR to their targets, in some cases to picomolar affinities
[18,19]. Generally, mutations in the TCR a and b CDR2 and
CDR3 regions can provide variants with enhanced pMHC
affinity either through direct contacts with the pMHC com-
plex or stabilization of the TCR in a optimal binding
conformation, though in some cases these variants also
show a loss in peptide specificity suggesting increased
interactions with the MHC component [20]. We and others
have found that certain CDR3 mutations can enhance spe-
cific binding of the scTCR to its cognate pMHC complex
and also improve interactions with epitope variants without a
general loss of peptide specificity [21]. Such enhanced
affinity TCRs have been found to useful in recognizing
viral targets known to evolve during infection to avoid
host T-cell immune responses. In the case of human immu-
nodeficiency virus (HIV) antigen-specific TCR, we have
isolated several different variants that bind most of the
known escape mutations for immunodominant epitopes
derived from vpr, p17, and p24 gag proteins. These reagents
have the potential to broadly recognize HIV-infected cells
carrying the HIV escape variants.
We have also employed a strategy for creating soluble
scTCR as multimers with increase avidity to target pMHC
molecules. In one example, STAR Multimer reagents have
been created that consist of a soluble scTCR containing
a biotin moiety. The biotinylated TCR molecules are
multimerized following addition to streptavidin carrying

31.2 EXPRESSION AND PURIFICATION
OF RECOMBINANT STAR FUSION PROTEINS

STAR fusion proteins are produced in a soluble, active and
fully glycosylated form by a recombinant Chinese hamster
ovary (CHO) cell expression system. Mammalian cell post-
translational modifications such as glycosylation are known
to provide improved pharmacokinetics and lower immuno-
genicity of therapeutic proteins. In addition, glycosylation at
particular sites in the TCR b chain may play a role in proper
folding of the molecule [25]. Successful expression of TCRs
of many different sequences clearly indicates that the
“single-chain” format permits functional folding without
the requirement of additional stabilizing modifications,
denaturation/refolding steps or specialized production cells.