Synthetic Biology Parts, Devices and Applications

17.3 Synthetic Biology Approaches to Cellular Immunotherapy Engineering 353

activity toward the target cell. Second- and third- generation CARs have further
incorporated costimulatory domains such as CD28 and 4-1BB to enhance T-cell
effector functions [55–58]. The modular nature of CARs is highlighted by the diver-
sity of targets that can be recognized by simply replacing the scFv while retaining
essentially the same transmembrane and cytoplasmic domains [59].
Although the first CARs predate the emergence of synthetic biology as a disci-
pline, CAR engineering is highly compatible with the synthetic biology approach
to biological system design and construction. Taking advantage of the modular
composition of CAR molecules, researchers have systematically probed the rela-
tionship between CAR structure and T-cell function by characterizing panels of
related CAR molecules [60], a process that has been greatly facilitated by the
advent of high-throughput DNA synthesis and assembly techniques. For exam-
ple, studies using a combinatorially constructed panel of CAR molecules have
demonstrated that the optimal length of the extracellular spacer in CARs is con-
tingent upon the size of the antigen presented by the target cell [61]. The effects
of adding different costimulatory signals and extracellular spacers to CARs have
also been explored by combinatorial cloning of CAR molecules [57, 62].
Beyond elucidating principle design rules, this bottom-up approach to CAR
engineering has been further leveraged to yield receptors that can execute
Boolean logic and regulate T-cell activation according to simultaneous or

Patient

blood

T-cell
isolation

T-cell

expansion

T-cell

reinfusion

T cell

Tumor-reactive

T cells

Reinfusion

product

Genetic

engineering

TIL

isolation

Tumor

biopsy

Figure 17.1 Schematic of adoptive T-cell therapy. Endogenous tumor-infiltrating lymphocytes
(TILs) are T cells with natural tumor reactivity and can be isolated from tumor biopsies,
expanded ex vivo, and reinfused into the cancer patient. Alternatively, non-tumor-reactive
T cells can be isolated, genetically modified to express a tumor-reactive T-cell receptor (TCR)
or chimeric antigen receptor (CAR), expanded ex vivo, and reinfused into the patient.