Biomedical Engineering Reference
In-Depth Information
As indicated earlier, this format facilitates genetic fusions
to other biologically active protein domains, allowing the
generation of the STAR-Ck, STAR-Ig, and STAR-Ab
reagents [5,14]. These advantages have allowed us to rapidly
develop a broad platform of scTCR-based reagents directed
against novel cancer and viral targets.
Generally, antigen-responsive human T-cell clones or
pMHC tetramer-sorted T-cell lines obtained from patients
with cancer or viral infections have served as the source to
generate cDNAs encoding disease antigen-specific TCR
a
and
b
chains [15,23]. We have also isolated TCR genes
from high-affinity T-cell clones obtained from peptide immu-
nized HLA-transgenic mice [5]. Additionally, following the
strategy of generating human antibodies in mice, we are
creating transgenic mice containing unrearranged human
TCR loci in the background of transgenic HLA alleles.
Successful generation and characterization of such mice
has recently been described by Li et al. [26]. This and our
studies confirm that transgenic human TCR
a
and
b
loci can
utilize mouse recombination enzymes and functionally
rearrange during T-cell development to generate a diverse
repertoire of human
a
/
b
TCRs [26]. Additionally, human
a
/
b
TCR loci transgenic mice carrying a HLA-A
0201 transgene
could be immunized with human tumor peptide antigens to
generate reactive T-cells with antigen-specific TCRs similar
to those obtained from cancer patients [26]. Because of
sequence divergence between human and mouse proteins,
the human
a
/
b
TCR loci transgenic mice likely are not
tolerized to most human tumor antigens allowing generation
of T cells to many target antigens through standard immuni-
zation methods [26]. It has previously been shown that HLA
transgenic mice lacking human CD4 or CD8 co-receptor
transgenes can be used to generate TCRs, which are co-
receptor independent and exhibit higher binding affinity
toward the cognate peptide/HLA complexes [27].
Once isolated by standard PCR-based methods, the
TCR
a
and
b
chain cDNA are constructed as scTCR genes
in a mammalian expression vector followed by transfection
into CHO cells. Short-term expression that permits assess-
ment of the secreted fusion protein levels and pMHC-
binding activity by standardized ELISAs using anti-TCR
C
b
Abs and pMHC tetramer reagents. These simple meth-
ods allow rapid identification of functionally paired
a
/
b
chains and initial evaluation scTCR affinity and produc-
tivity. Suitable fusion proteins are then selected for larger
scale production in stably transfected CHO cell lines in
suspension culture conditions, a standard method for ther-
apeutic protein manufacture. Using these methods, pro-
duction of scTCR/IgG1 fusion proteins can reach the
0.5 g/L level, comparable to that observed for structurally
similar antibody molecules. Further improvement in
expression to the gram per liter level is expected to be
highly feasible to support late phase clinical development
or product commercialization.
The STAR fusion proteins are then purified via affinity
chromatography using anti-TCR C
b
antibody or protein A
(forscTCR/Igfusion)coupledresinfollowedbyanion
exchange chromatography step [5,14,15,17]. For GMP
production of clinical product, robust viral inactivation
and removal steps (i.e., low pH load, viral filtration) are
also included in the purification process. The final product
is formulated in a buffered saline solution and extensively
characterized for composition, purity, and potency (i.e.,
TCR binding activity and effector domain function) using
validated assays. In such a formulation, the STAR-Ck final
vialed product (ALT-801) is stable for at least 4 years
when stored at 5
C. As a result, the “industry-standard”
upstream and downstream production methods developed
for these fusion proteins are well suited to support late
stage clinical development and potentially therapeutic
product commercialization.
31.3 CLINICAL AND RESEARCH PRODUCT
APPLICATIONS
As indicated earlier, we are utilizing the novel STAR
platform technology to capture the precise, disease-targeting
properties of TCRs in creating next-generation targeted
therapeutic molecules for cancer and viral infection. Addi-
tionally, STAR reagents are being developed for diagnostic
applications to identify disease-associated antigens derived
from intracellular proteins as biomarkers [15] and for
research application to better understand antigen presenta-
tion and T-cell immunology [28,29].
31.3.1 Cancer Therapeutics
Our strategy is to develop scTCR-based targeted therapeu-
tics against broad-based tumor-associated antigen. Our
lead anticancer agent, ALT-801, is a recombinant soluble
scTCR/IL-2 cytokine fusion protein that recognizes cancer
cells through p53-derived targets. Human p53 tumor sup-
pressor protein is a particular interest for cancer target as it is
mutated and overexpressed in up to 50% of all human
cancers [30]. Normally, this protein that acts to arrest the
proliferation of cells in response to various stresses, includ-
ing DNA damage, hypoxia, and cell cycle aberrations [31].
When mutated, it loses its ability to suppress abnormal
proliferation and accumulates within the cells [31]. As a
result, p53 mutation and overexpression in cancer cells
correlates with transformation events and tumor aggression
and is associated with lower overall survival rates and
resistance to chemotherapeutic intervention [32]. Since
only low amounts of native p53 are detectable in normal
tissue, the differential display of p53 in tumor versus normal
tissues creates the opportunity to therapeutically target this
protein. However, p53 is an intracellular protein and is not
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