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Page 81 Cox et al. J Transl Genet Genom 2021;5:80-8 https://dx.doi.org/10.20517/jtgg.2021.06

INTRODUCTION
Over the last five years, chimeric antigen receptor T (CART) cell therapy has emerged as a new therapeutic
[1]
strategy in the treatment of diseases . In addition to surgery, chemotherapy, and radiation, CART cell
therapy has become a fourth pillar in the treatment of cancer . Although the first FDA approval of a CART
[2]
[3,4]
product was achieved only in 2017 , the concept of a chimeric antigen receptor (CAR) was first pioneered
[5]
by Gross et al. in 1989, and the latest clinical successes are the result of decades of incremental scientific
advancements in the fields of immunology, molecular biology, and tissue culture. A CAR is a recombinant
protein composed of the single chain variable fragment from a monoclonal antibody, which is fused to a
transmembrane domain and the intracellular signaling domains of the T cell . The first design contained a
[6]
single CD3ζ signaling domain, and while it showed promise in vitro, the clinical activity was minimal . The
[6]
second generation, now-FDA-approved CART cells use CD3ζ coupled with an additional co-stimulatory
[7]
domain (e.g., 4-1BB or CD28) .
The process of generating CART cells is complex. First, patients’ peripheral blood mononuclear cells are
harvested through a process called apheresis. Next, their isolated T cells are stimulated ex vivo to expand T
cells and introduce the CAR transgene. Following transfection, T cells are expanded for 1-2 weeks before
they are reintroduced into the patients. This process takes 3-4 weeks from apheresis to reinfusion of CART
[1]
cells back into patients .

Over the last decade, CD19-directed CART cell (CART19) therapy has yielded unprecedented outcomes in
patients with B cell malignancies. In patients with relapsed, refractory B-acute lymphoblastic leukemia
(ALL) where prognosis is dismal, a single treatment with CART19 therapy resulted in a complete remission
of 80%-90% of patients and durable remissions beyond 2 years in 50%-60% of patients, highlighting the
[3,8]
curative potential of the therapy . Similar results were achieved in lymphoma and multiple myeloma,
leading to the FDA approval of five different therapies thus far [9,10] .

Despite the success and FDA approval of multiple CART cell therapies, its wider application is limited by
the development of resistance and life-threatening toxicities. While the initial response rates to CART19 in
B cell malignancies are high, most patients relapse within the first two years [10,11] . Moreover, CART cell
activity in solid tumors is very limited and objective responses are rarely seen . While CART cells are
[12]
found at the tumor site in solid tumor models such as glioblastoma and mesothelioma, the T cells seem to
be rendered dysfunctional by the tumor microenvironment (TME) [13-15] .

The administration of CART cell therapy is also associated with life-threatening toxicities, namely cytokine
release syndrome (CRS) and/or neurotoxicity . CRS is related to extreme elevation of cytokines correlating
[1]
with T cell proliferation in vivo and presents as hypotension and respiratory failure, occasionally leading to
[1]
deaths . The pathophysiology of neurotoxicity is not well understood, but mechanistic studies point toward
a role for myeloid cell activation and their cytokines in the development of neurotoxicity .
[1]
A comprehensive understanding of the phenotypical, functional, and molecular state of T cells, tumor, and
TME would allow (1) the identification of predictive biomarkers to identify non-responders and patients at
risk of developing life-threatening toxicities; and (2) the development of strategies to overcome resistance
and toxicities. Collectively, this suggests that the major limitations to CART cell therapy include: (1)
resistance and inhibition; (2) the development of life-threatening toxicities; and (3) lack of predictive
biomarkers for response and toxicities.