Biomedical Engineering Reference
In-Depth Information
means to display and specifically recognize a wide range of
peptide antigens derived from infection agents or malignant
cells that threaten the survival of the host. Consequently,
there is considerable interest in developing TCR-based
agents to target this distinct class of antigens, particularly
those derived from intracellular proteins, which constitute a
majority of the disease-relevant antigens [3,4].
mammalian cells [5,14,15], insoluble/refolded proteins from
bacterial cells [12], and coat protein fusions displayed on the
surface of bacteriophage [16]. In each case, the scTCR
retained the ability to specifically recognize the appropriate
cognate pMHC complex. Additionally, inclusion of the
invariant TCR C
b
domain provides a convenient means
of purifying scTCR molecules via immunoaffinity chroma-
tography using anti-murine or human TCR C
b
antibodies
[5,14,15,17]. We have used this three-domain format to
express soluble scTCRs with over 30 different pMHC
specificities and hundreds of different unique scTCR var-
iants confirming the broad utility of this approach for
generating functional TCR molecules with different
sequences.
Another limitation with TCR-based targeting is the fact
that soluble TCRs per se do not possess any functional
effector activity and binding of TCRs to pMHC complexes
does not induce target cell death. As a result, we have
developed a fusion protein approach to link the scTCR
domain to molecules such as cytokines (STAR-Ck), immu-
noglobulin constant regions (STAR-Ig), and single-chain
antibodies (STAR-Ab) (Figure 31.1). Such genetic fusions
are made to the C-terminal TCR C
b
region of the scTCR
which is distal to the TCR-binding domain and does not
impact interaction between the scTCR and its cognate
pMHC target. In each of the fusions, the scTCR component
functions to guide and deliver the therapeutic agent or
effector function to the disease site where the cytokine or
antibody binding/effector domains stimulate and augment
an immune response. Using a similar approach, we have also
generated scTCR-conjugated imaging agents and therapeu-
tic drugs. Current development is focused on improving the
activities and lowering the systematic toxicities of FDA-
approved agents such as interleukin-2 (IL-2) (Proleukin
1
),
GM-CSF (Leukine
1
), and doxorubicin, and promising drug
candidates, such as IL-15, for treatment of malignant
31.1.1 Three Domains Single-Chain TCR (scTCR)
Format and scTCR-Fusions
Despite structural similarities with extracellular antibody
domains, production of soluble recombinant TCRs has
proven difficult. The main technical limitations are low
expression yields and instability of individual
a
or
b
chains
and unstable
a
/
b
pairing in the absence of anchoring
transmembrane domains and/or interchain disulfide bonds.
To overcome these issues, a single-chain approach, similar
to that used with single-chain (sc) Fv antibody domains, has
been explored where the TCR V
a
and V
b
domains are
covalently linked by a flexible peptide sequence [5-8].
While initial attempts to effectively generate soluble TCR
V
a
-V
b
single-chain molecules were not generally success-
ful [6-8], subsequent structural studies showed that a prom-
inent elbow region at the N-terminus of the C
b
domain
makes numerous contacts with the V
b
domain, suggesting
co-expression of these domains may promote for proper
folding of soluble protein [9]. In contrast, less extensive
interactions between the C
a
and V
a
domains are consistent
with observations that the C
a
domain was dispensable for
expression of individual V
a
chains or scTCR molecules
[10-13]. Based on these finding, we and others have
employed a three domain V
a
-linker-V
b
-C
b
format to
express scTCR molecules [5,12-16]. This three domains
scTCR format has been successfully used to generate mem-
brane-bound proteins [13] or soluble-secreted proteins from
FIGURE 31.1
Diagram of STAR fusion proteins.
Search WWH ::
Custom Search