Biomedical Engineering Reference
In-Depth Information
aspect to note is t hat the potenti al appl icabili ty of a T CR to
a patient population i s defined by the prevalence of both
the present ing MH C allele and of the antigenic protein
f r o m w h i c h t h e p e p t i d e a n t i g e n i s d e r ived . M H C , o r H L A ,
types differ considerably i n t heir overall prevalence and
ge ographical distribution. HLA-A2, for example, is found
in
together (Figure 32.2A). Many strategies have been tried
to produce a soluble TCR framework. An scFv-like single
chain design, offering the advantage of a single gene
expression, might appear to be the simplest approach and
single chain versions of some TCRs, using various designs
[10], have been produced. However, the strategy is not
generally applicable and the stability of conformation
between the two variable domains is uncertain. For achiev-
ing maximal structural stability and integrity, as well as for
avoiding immunogenicity, an authentic heterodimer com-
prising the two chains, truncated downstream of the native
interchain disulfide, provides a more dependable solution.
Such designs have been tried, but found unworkable, due to
aggregation of chains, probably caused by incorrect
disulfide bond formation in the process of protein folding.
An alternative design, in which fos and jun leucine zippers
were fused C-terminally to the truncated chains [11], pro-
vided good stability of the heterodimers and was success-
fully applied to all tested TCRs (Immunocore, unpublished
data). For therapeutic applications, however, such a protein
design would be too unstable. Instead, with the aim of
generating a simple, generally applicable and highly stable
TCR framework, several locations within the constant
domains were investigated by molecular modeling for the
introduction of a new interchain disulfide link [12]. Several
suitable locations were identified, the theoretically optimal
one being between TRAC threonine 48 and TRBC serine
57, which were therefore mutated to cysteines. Expressed
separately in Escherichia coli, the chains were refolded in
vitro, resulting in a highly stable, fully human soluble
protein, which became known as the monoclonal TCR
(mTCR). A large number of individual TCRs of varying
peptide specificity, as well as HLA restriction, have been
successfully produced as mTCRs, establishing this as the
optimal generic soluble TCR format for crystallographic
studies as well as for potential therapeutic uses.
The crystal structure model of a tumor-associated NY-
ESO-1 peptide-specific 1G4 mTCR [12] docking onto its
cognate peptide-HLA-A2 is shown in Figure 32.2B. The
TCR's ligand-binding surfaces, in an analogous fashion to
those of antibodies, are formed by six complementary
determining regions (CDRs), which are hyper-variable
loops protruding from the membrane-distal end of the
variable domains. Counting from the N-terminus of each
chain, these are called CDR
a
1,
a
2,
a
3and
b
1,
b
2,
b
3,
respectively. While the amino acid sequences of CDRs 1
and 2 are determined in the germ line and thus are fixed
in each type of variable domain (of which there are 54
a
and 67
b
sequences in humans), the CDR3s are formed as
a result of gene recombination, and consequently unique
for each T-cell clone. Despite the significant variations in
the docking mode observed in crystal structures of
TCR/peptide-HLA complexes [12-18], one characteristic
feature is that
50% of the European and North American populations
and
30% of A sian populations. The equivalent preva-
lence of HLA-A24 in the same populations is 18 and 65%,
r e s p e c t ivel y.
1
While H LA type limits the proportion of
patients that can be targeted w ith a specific TCR, the
a l m o s t u n iver s a l c h o i c e o f , p a r t i c u l a r l y i n t r a c e l l u l a r, a n t i -
gens means that peptide-MHC antigens can offer better
patient coverage than other c ell -surface antigens such as
thos e t ha t c an be targeted by antibodies. Her2-neu a s a n
antibody targ et in breast cancer, f or example, is present in
22% of the patient population.
2
Some intracellular
antigens, in contra st, a re present at very high f re quenci es
in certain t um or types. Gp100, a protein expressed i n
vi rtually 100% of malignant melanomas, is one such
example [ 4]. A TCR r ecognizing gp100 presented by
HLA-A2 can therefore target
50% of patients in Europe
and North America. IMCgp100, a high-affinity gp100
specificTCRfusedtoananti-CD3scFv,hasentered
cli ni cal trials in metast at ic melanoma both t he United
Kingdom and the U ni ted St ates in 2010.
32.2 ENG INEERE D HIGH -AFF INITY
MONOC LONAL TCR s (mTCR )
Fr om a protein engineering perspective , an immediate
advantage is that, in contrast to t he anti gens bound by
antibodies, peptide-MHC c om pl exes a re easily produced
by a s ta nd a rd in vi tro refolding procedure. The peptide of
interest i s synthesized, com bined with the c orresponding
MHC type polypeptide a nd
b
2m and purified after refold-
ing [5]. TCRs t hemselves diffe r from a nt ibodi es in t wo
important characteristics. First , TCRs do not nat ural ly
exist as s oluble, noncell-bound molecules. Second, the
affini ties of n at urally occurr ing TCRs to their correspond-
ing peptide-HLA ligands fall i nt o a narrow range of about
1-300
m
M[6-9],whichistoolowforthemtobeusedin
any therapeutic or diagnostic ap plications. Structurall y, the
extracellular par t of a TCR resembles an antibody Fa b
fragment, w ith its sim ilarl y s iz ed al pha a nd be ta c h ai ns ,
ea ch c o mpri si ng a m em br ane -dis ta l N -te rm inal var ia ble
domain and a membrane-proximal constant domain, with
an intrachain disulfide bond s tabilizing each of the four
domains. An interchain disulfide bond, l ocated near the
cell surface in the constant domains, links t he two c ha ins
1
http://www.allelefrequencies.net/hla.
2
Stakeholder Insight, Datamonitor 2010.
the CDR3s of both chains only make
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