Biomedical Engineering Reference
In-Depth Information
TABLE 32.2 Examples of mTCR Affinity Maturation
With the indirect approach, the protein fused to the
mTCR must convey a potent activation signal to the immune
system, for example, T cells, in the vicinity of the target
cells. Such a protein moiety could be a cytokine, a receptor, a
bacterial superantigen, or an antibody fragment. Many such
possibilities have already been tested on antibody fusions.
With this approach also, the potency of the fused effector
function is the key property. The cytokine interleukin-2
(IL-2), fused to a tumor-specific, high-affinity NY-ESO
mTCR, showed some promise in stimulating CD8
Specificity
k
a
k
d
K
D
T
1/2
4.0
10
4
6.6
10
5
1.3
10
1
1.7
10
5
NY-ESO
32
m
M
26 pM
7s
19 h
MAGE A3
ND
3.4
10
5
ND
2.2
10
5
281
m
M
58 pM
ND
9h
2.8
10
3
7.5
10
4
1.1
10
1
4.9
10
6
Telomerase
38
m
M
65 pM
3s
39 h
gp100
ND
5.3
10
5
ND
5.8
10
6
26
m
M
11 pM
ND
33 h
cytotoxic
T lymphocytes (CTLs) to exhibit activity against NY-ESO
peptide-pulsed targets but, considering the toxicity of IL-2
versus the efficacy that could be achieved, this option was not
considered attractive as a potential therapeutic (unpublished).
An alternative way of addressing the potency problem
would be through increasing the payload, that is, the number
of effector function moieties conjugated to each mTCR
molecule. However, the opportunities for generating direct
genetic fusions, which are suitable for larger scale manufac-
turing purposes, are limited. Although four potential fusion
points exist on mTCRs, counting the N- and C-termini of the
ab
heterodimer (Figure 32.2), only one is typically used, due
to aggravating expression and folding problems. Tandem
effector function molecules [28], while in theory attractive,
in many cases have limited applications in practice for the
same reasons. The future here may lie with nongenetic
conjugates, involving newly developed protein nanoparticles
as well as dendritic polymers and possibly other advanced
delivery systems.
þ
WT1
ND
9.9
10
5
ND
1.6
10
5
96
m
M
16 pM
ND
12 h
For each specificity, wild type: top, affinity enhanced: bottom (ND: not
determined due to kinetics too fast for measurements).
32.3 mTCR-BASED FUSION PROTEINS
FOR THERAPEUTIC APPLICATIONS
Unlike antibodies, mTCRs do not encompass an Fc frag-
ment or any other effector function and targeted cell
killing, therefore, requires conjugation or fusion to a
suitable cytotoxic mediator. Because any particular
tumor-associated, peptide-MHC antigen is presented at
very low copy numbers per cell, typically in the range
10-150, the effector function must have very high cyto-
toxic potential. This rules out some of the conjugates used
for therapeutic antibodies, for example, radioactive label-
ing, which requires several thousand targets per cell to
achieve a cytotoxic effect. Bearing this difference in mind,
the strategies for choosing an effector function to be linked
to mTCRs can follow similar paths to those explored with
antibodies. The two main modes of action that can be
employed are (a) a direct mechanism delivering a payload,
for example, a cytotoxin, to the cancer cell surface, or (b)
an indirect mechanism whereby immune activity is redir-
ected against the cancer cell.
The first approach involves conjugating or fusing a cell
toxin molecule to the mTCR. As no amplification of the
killing mechanism can be achieved and the antigen target
number per cell is very low, the toxin needs to be of very
high potency, even in a situation where high mTCR affinity
enables almost saturating binding to the antigen. While a
single molecule of the Pseudomonas exotoxin A-derived
PE38 is, in theory, sufficient to kill a eukaryotic cell, this is
not achievable in practice, probably because the toxin
requires internalization and mTCRs, when bound to antigen,
largely remain on the cell surface (unpublished). PE38-fused
mTCRs were found to be capable of killing peptide-pulsed
target cells (which can present nonphysiologically
high numbers of antigen per cell), but were not potent
enough for efficiently destroying naturally presenting tumor
cells (unpublished).
32.4 IMMUNE-MOBILIZING MONOCLONAL
TCRs AGAINST CANCER (ImmTAC)
Probably, the most potent cytotoxic antibody-based fusions
reported to date are the so-called BiTEs, which comprise a
targeting antibody fragment linked with a T-cell-activating
anti-CD3 scFv antibody [29-31]. A series of fusion proteins,
consisting in each case of a high-affinity cancer-specific
mTCR and an anti-CD3 scFv, have been produced and
optimized. The TCR
a
and TCR
b
-scFv chains comprising
these fusion molecules, called ImmTACs, are expressed
separately at extremely high levels in E. coli. The ImmTAC
is then generated by mixing and refolding the two chains,
which recovers a yield in excess of 10% and produces protein
that is homogeneous and, by release assays, 100% active.
Functionally, the mTCR part of the fusion binds to an
antigenic peptide-MHC on the target cell, while the other
end of the molecule, by engaging the
-chain of the CD3
glycoprotein, is capable of activating any CD8
e
CTL,
independently of the intrinsic antigen specificity of the
latter (Figure 32.4). Several of these CTL-activating agents
have been comprehensively tested in cellular assays with
promising results. Figure 32.5 shows the properties of one
þ
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