356 17 Synthetic Biology in Immunotherapy and Stem Cell Therapy Engineering
The modular nature of CAR signaling has also spurred the development of a
number of dual-CAR systems that trigger T-cell activation only if two conditions
are simultaneously satisfied, in essence executing AND-gate or AND-NOT-gate
computations that aim to improve targeting specificity by therapeutic T cells.
In one example, researchers designed inhibitory chimeric antigen receptors
(iCARs) by replacing the CD3ζ domain of a prostate-specific membrane antigen
(PSMA)-targeting CAR with intracellular signaling domains from inhibitory
receptors such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and
programmed death 1 (PD-1) (Figure 17.2c) [69]. Upon recognition of PSMA,
inhibitory signaling through the iCAR effectively competed against activating
signaling by a second-generation CD19 CAR to limit T-cell proliferation,
cytokine secretion, and cytotoxicity, thereby achieving “CD19-AND-NOT-
PSMA” signal computation [69]. It is important to note that NK cells naturally
express both activating and inhibitory receptors, and insights to be gained from
greater understanding of NK cell signaling may also serve to instruct the robust
development of iCARs.
Building on the clinical observation that both the CD3ζ chain and costimula-
tory signals are necessary to achieve in vivo antitumor responses, another study
described a dual-receptor system in which the first receptor targets the prostate
stem cell antigen (PSCA) and contains only the CD3ζ chain without costimula-
tory signals, while the second is a chimeric costimulatory receptor that targets
PSMA and contains both CD28 and 4-1BB costimulatory signals but no CD3ζ
chain (Figure 17.2d). After testing several anti-PSCA scFv domains with varying
binding affinities, researchers were able to generate a pair of receptors that
trigger T-cell activation and effectively control tumor growth in vivo if and only
if the tumor expressed both PSCA and PSMA [70]. An interesting alternative
approach is to segregate the extracellular scFv from the CD3ζ chain until
re constitution via small molecule-induced heterodimerization. A recent study
reported the construction of ON-switch CARs by incorporating the rapamycin
analog (rapalog)-inducible heterodimerization domain FK506 binding protein
(FKBP) into a truncated second-generation CD19 CAR lacking the CD3ζ chain.
Separately, the FKBP-rapamycin binding (FRB) domain was fused to the cyto-
plasmic portion of CD3ζ (Figure 17.2e) [71]. As such, a fully functional CAR
containing the ligand-binding scFv domain and the T-cell-activating CD3ζ chain
is only generated upon the addition of rapalog, which induces dimerization
between FKBP and FRB, thus bringing the two system components into close
proximity. T cells expressing the ON-switch CAR were able to proliferate and
mediate cytotoxicity upon target-cell encounter, but only in a rapalog dose-
dependent manner, thus yielding temporal control over CAR activation via small
molecule drug administration [71].
Yet another example of synthetic receptor design repurposes the signaling
mechanism of the Notch receptor. Antigen binding by the Notch receptor
exposes a juxtamembrane cleavage sequence that undergoes proteolysis by the
intramembrane protease gamma-secretase, a processing step that releases the
intracellular Notch domain to the nucleus to serve as a transcription factor (TF)
that drives gene expression programs. Utilizing a modular design approach anal-
ogous to CAR engineering, researchers developed synthetic Notch (synNotch)