17.3 Synthetic Biology Approaches to Cellular Immunotherapy Engineering 357receptors comprised of an extracellular scFv domain fused to a synthetic TF via
the endogenous Notch transmembrane domain and juxtamembrane cleavage
sequence [72] (Figure 17.2f ). Upon ligand binding, the synNotch receptor under-
goes cleavage and releases the synthetic TF to drive gene expression from a
cognate inducible promoter. By placing CAR expression under this transcrip-
tional control, a dual-receptor system enables T cells to perform AND-gate com-
putation in a sequential manner – that is, antigen A triggers the synNotch
receptor to drive expression of the CAR, and subsequent recognition of antigen
B by the CAR activates T-cell effector functions. Pairing a green fluorescent pro-
tein (GFP) synNotch with a CD19 CAR enables T cells to effectively eliminate
tumor cells expressing both GFP and CD19, but not CD19 alone [73].
Although these strategies underscore the vast potential of applied synthetic
biology toward enhancing therapeutic efficacy and specificity, CAR performance
is still subject to design rules that require better understanding. Multiple recent
studies have implicated important CAR components, such as the framework
region of scFv domains and the non-signaling extracellular spacer, in triggering
tonic signaling [74, 75]. Moreover, in each of the examples highlighted previ-
ously, multiple iterations of receptor design were required to identify the correct
combination of “modular” components to achieve robust system performance.
As more data become available through systematic studies of CAR design param-
eters, a more quantitative, rational approach to next-generation CAR design will
begin to supplant what has largely been a trial-and-error method in engineering
CAR-T cells for disease treatment.
17.3.2 Genetic Engineering to Enhance T-Cell Therapeutic Function
Robust proliferation and persistence of T cells have been shown by multiple clin-
ical trials to be both critical to therapeutic efficacy and difficult to achieve in vivo
[32, 57, 58]. Consequently, there have been many attempts to prolong the sur-
vival of CAR-expressing T cells via genetic engineering. These approaches can
be broadly grouped into strategies that promote immune stimulation and those
that counteract immune suppression. Within the former category, researchers
have engineered “armored” T cells to overexpress immunostimulatory cytokines
including IL-2, IL-12, and IL-15, thus sustaining T-cell proliferation and effector
function [76, 77] (Figure 17.3a). Transgenic expression of costimulatory mole-
cules such as 4-1BB ligand (4-1BBL) and CD40L or surface receptors including
interleukin-7 receptor α (IL-7Rα), CCR4, and CXCR2 has also been shown to
mitigate T-cell exhaustion and promote T-cell persistence [78–82].
Even when armored with supportive cytokines and costimulatory signaling,
engineered T cells can still become exhausted or rendered dysfunctional by
repeated antigen stimulation or sustained exposure to immunosuppressive fac-
tors located in the tumor microenvironment. To overcome this challenge, exten-
sive research has focused on disrupting endogenous inhibitory signaling
pathways (Figure 17.3b) or rewiring immunosuppressive inputs to immunostim-
ulatory outputs (Figure 17.3c). Notably, clinical administration of monoclonal
antibodies targeting inhibitory checkpoint molecules such as CTLA-4 and PD-1
has been shown to alleviate immunosuppression of naturally tumor-infiltrating