REVIEW

Chimeric Antigen Receptor www.biotechnology-journal.com

Cancer Immunotherapy Using CAR-T Cells: From the

Research Bench to the Assembly Line
Diogo Gomes-Silva* and Carlos A. Ramos

Adoptive T-cell therapy (ATCT) is a form

The focus of cancer treatment has recently shifted toward targeted therapies, of immunotherapy that involves the isola-
including immunotherapy, which allow better individualization of care and tion of lymphocytes with the intent to
are hoped to increase the probability of success for patients. Specifically, T stimulate and expand ex vivo potent anti-
gen-specific T cells that are subsequently
cells genetically modified to express chimeric antigen receptors (CARs; CAR-T
infused into a patient to treat a disease.
cells) have generated exciting results. Recent clinical successes with this Adoptively transferred T cells are a “living”
cutting-edge therapy have helped to push CAR-T cells toward approval for drug that has potential advantages over
wider use. However, several limitations need to be addressed before the conventional therapies: T cells recognize
widespread use of CAR-T cells as a standard treatment. Here, a succinct tumor antigens through their antigen
receptor (T cell receptor, or TCR) allowing
background on adoptive T-cell therapy (ATCT) is given. A brief overview of
them to mount a strong specific immune
the structure of CARs, how they are introduced into T cells, and how CAR-T response potentially capable of eliminating
cell expansion and selection is achieved in vitro is then presented. Some of tumors in a short period of time and, in
the challenges in CAR design are discussed, as well as the difficulties that addition, are able to proliferate and poten-
arise in large-scale CAR-T cell manufacture that will need to be addressed to tially survive in vivo for years,[1] possibly
achieve successful commercialization of this type of cell therapy. Finally, granting them the ability to control tumors
that relapse. The most exciting results with
developments already on the horizon are discussed.
ATCT have been achieved by the genetic
modification of Tcells with chimeric antigen
receptors (CARs; CAR-T cells). Recent clini-
cal successes with this cutting-edge technol-
1. Introduction ogy have helped to push T cell therapy toward approval for wider
use. However, several limitations need to be addressed before the
Surgery, chemotherapy, and radiation therapy, alone or in
widespread use of CAR-T cells as a standard treatment.
combination, have been the mainstay of cancer treatment.
Here, we will give a succinct overview of the structure of
Together with newer and more accurate diagnostic tools, these
CARs, how they are introduced into T cells, and how CAR-T cell
approaches have contributed to substantially improved out-
expansion and selection is achieved in vitro. We will provide a
comes. However, the prognosis of most malignancies remains
basic description of CAR design and clinical applications. We
poor. Given their complexity, most cancers will ultimately
will mention some of the challenges in CAR design, and some of
require more personalized management to achieve cure or
the difficulties that arise in large-scale CAR-T cell manufacture
control. Recently, the focus of cancer treatment has shifted
that will need to be overcome to achieve successful commercial-
toward targeted therapies, including immunotherapy, which
ization of this type of cell therapy. Finally, we will discuss
allow better individualization of care and are hoped to increase
developments already on the horizon.
the probability of success for patients.

D. Gomes-Silva, Dr. C.A. Ramos

2. Chimeric Antigen Receptors (CARs)
Center for Cell and Gene Therapy, Baylor College of Medicine Some of the limitations seen with earlier types of ATCT can be
Texas Children’s Hospital and Houston Methodist Hospital
Houston, TX 77030, USA
overcome by redirecting T cell activity toward an antigen using a
E-mail: diogos@bcm.edu recognition system that relies on the antigen binding abilities of an
D. Gomes-Silva antibody molecule. A CAR is a synthetic molecule that does just that.
Department of Bioengineering and In brief, a CAR is a protein that fuses an extracellular, antibody-
iBB—Institute for Bioengineering and Biosciences, derived antigen recognition domain with intracellular TCR-derived,
Instituto Superior T
ecnico activating domains[2] (Figure 1). The extracellular binding moiety
Universidade de Lisboa,
Lisboa, Portugal
provides the antigen specificity and is commonly a single-chain
fragment variable (scFv) originating from a monoclonal antibody.
The ORCID identification number(s) for the author(s) of this article When T cells are engineered to express a CAR, after binding the
can be found under https://doi.org/10.1002/biot.201700097.
target antigen via the scFv, they get activated through the signaling
DOI: 10.1002/biot.201700097 components included in the CAR.

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Diogo Silva graduated in Genetics

and Biotechnology and has a Master
in Biomedical Research. Currently he
is finishing his Doctoral program
from the University of Lisbon,
Portugal, in collaboration with the
Baylor College of Medicine, USA, in
CAR-T cell therapies, and is involved
in multiple projects for the
generation of several CARs to be
used against different hematologic tumors.

Dr. Ramos earned his Medical

Degree from the University of
Lisbon, Portugal, and was a Fellow
in Hematology and Medical
Figure 1. Schematic representation of a Chimeric Antigen Receptor Oncology at the Memorial Sloan-
(CAR). CARs currently in clinical use have an extracellular antigen
Kettering Cancer Center, in New
recognition domain (here represented by an antibody-derived single-
chain variable fragment, scFv), one or more co-stimulatory domains
York, and a Fellow in Blood and
(signal 2), and CD3ζ (signal 1). Marrow Transplantation at M. D.
Anderson Cancer Center, in
Houston. He is currently Associate
Since CARs provide MHC-independent antigen recognition, Professor of Medicine in the Center for Cell and Gene
some of the mechanisms used by tumor cells for immune escape, Therapy at the Baylor College of Medicine, in Houston.
such as downregulation of MHC molecules, can be mitigated;[3] in His research focuses on T-cell therapy for cancer and he
addition, a CAR specific for a certain antigen could be used in is the principal investigator in phase 1 trials of CAR-T
patients regardless of their specific HLA type (a common cells targeting CD19, CD30, and к light chain as therapy
limitation of other engineered T cells). Another advantage of for lymphomas.
CAR-mediated antigen recognition is that antigens other than
proteins, such as carbohydrates and lipids, can be recognized.[4,5]
Nonetheless, CAR-T cells have some drawbacks. Whereas
traditional T cell responses through TCR and MHC interaction 2 clinical trials of CD19 CAR-T cells have consistently shown
can recognize intracellular proteins (which account for more high antitumor efficacy, with complete response rates of 70–90%
than 90% of potential tumor-associated antigens, or TAAs) that in pediatric and adult patients with relapsed or refractory,
are processed and presented by the MHC molecule, binding of chemotherapy-resistant acute B-cell leukemia. These encourag-
the CAR is limited to molecules that are present on the surface of ing results have attracted the interest of pharmaceutical and
tumor cells. Another potential disadvantage is that, because scFv biotech companies and a CD19 CAR-T cell product was recently
sequences are usually derived from murine proteins, humoral approved by the FDA for commercial use outside clinical trials by
and cellular immune responses can occur against this portion of Novartis Pharmaceuticals Corp. (KymriahTM), and more com-
the molecule and lead to elimination of CAR-T cells from panies are likely to see their products approved in the next
circulation. This problem, which has been previously docu- months. Meanwhile, CAR-T cells directed against other disease
mented in some patients, may be minimized by using scFv targets, both in hematological (such as BCMA,[10] CD20,[11]
whose amino acid sequences have been fully humanized.[6] CD33,[12] or CD123)[13] and solid tumors (such EFGRvIII,
Although early (first generation) CARs contained only one GD2,[14] and Her2),[15] are now being tested in clinical trials.
activating domain (usually a portion of the ζ chain in the TCR
complex) and thus mimicked only TCR activation (signal 1), all
clinically effective CARs include extra activating domains that
3. CAR Optimization
trigger additional stimulatory pathways (signal 2). Most
commonly, the costimulatory domains included are CD28 or Despite encouraging clinical results with CD19 CAR-T cells,
4-1BB (CD137). These second (with one additional activating there is considerable heterogeneity in the structure of CARs
domain) and third (with two or more additional domains) (reviewed elsewhere)[16] used across trials in different institu-
generation CARs are associated with increased CAR-T cell tions. Yet, it is unclear whether one type of CAR has advantages
proliferation and cytokine production both in preclinical models over another, even when targeting the same antigen. CARs have
and in clinical trials.[7] been designed empirically; distinct backbones, types and
While CAR-T cells can in theory be redirected against any number of signaling endodomains, and methods of CAR
surface-expressed target of choice, most clinical trials so far have delivery into T cells likely affect antitumor activity. For instance,
been directed at CD19, an antigen that is expressed in B-cell the spatial interaction between a cancer cell and a T cell depends
malignancies (reviewed elsewhere).[8,9] For example, phase 1 and on the exact location of the epitope being recognized on the

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target molecule and the structure of the extracellular domain of expression of the CAR is transient, this method may be optimal
the CAR. Differences in T cell physiology and activation owing to in instances where persistent targeting of an antigen may be
the use of distinct scFvs recognizing the same antigen, different associated with life-threatening toxicities. Clinical trials of
co-stimulatory endodomains[17] or different lengths and sequen- mesothelin-specific CAR-T cells transduced using this method
ces of the CAR extracellular domain[18] have been documented. are now in progress (NCT01355965) to investigate if CAR
While it is conceivable that there is an optimal distance and angle expression will persist long enough to elicit antitumor effects but
of interaction between CAR-T cells and target cells for proper T short enough to prevent side effects.
cell activation, these properties are often impossible to predict. The most recent approach to genetically modified T cells uses
Therefore, although the backbone of the CAR and the number gene editing tools such as CRISPR or TALEN to create a double
and type of its endodomains are an absolute determinant of the break in a particular DNA site, with the CAR gene being
overall antitumor effect observed in patients, their optimization is provided as a DNA template that is introduced into that selected
currently a laborious and trial-and-error procedure, with multiple genomic site. This system minimizes the risk of unrestrained
iterations of “bench to bedside and back again” approaches. This genomic integration and has demonstrated good antitumor
process makes translation to the clinic difficult and time effect in a preclinical model.[26] It is, however, more complex
consuming. It is clear, however, that the CAR design process than other approaches. Nonetheless, the use of this engineering
does not follow a “one size fits all” model and that each CAR must system is under intense study.
be designed, optimized, and tested for the specific target molecule Regardless of the actual method used for Tcell transduction, from
in order to generate the ideal CAR for a particular tumor. a biotechnology point of view, all these vectors can be made in large
quantities and frozen for several years. When Tcells are available for
transduction, an aliquot can be thawed and used as needed.
4. Engineering the T Cell to Express a CAR
After a CAR is designed, its gene needs to be permanently
5. CAR-T Cell Manufacturing
introduced into T cells. The most commonly used vectors for this
purpose are replication-defective retroviruses of two types: γ- The first step in the generation of autologous CAR-T cells is the
retrovirus and lentivirus. Both have an RNA genome that is reverse- collection of peripheral blood mononuclear cells (PBMC) from a
transcribed into DNA after T cell transduction, which then gets patient, usually through leukapheresis, whereby blood is
integrated into the host genome. This integration process raises removed from the donor and separated into different compo-
concerns about potential insertional mutagenesis,[19] to which nents, with the immune cells being collected while all other
lentivirus may be less prone by favoring insertion sites away from components returned to the donor’s circulation. T cells are then
active genes.[20,21] Factors such as the transgene and its promoter, cultured in the presence of a nonspecific Tcell stimulus, which is
and the cell type being transduced also seem to influence the risk of required not only for expansion but also for efficient genetic
mutagenesis.[22] T cell transduction appears to be safe especially modification, since most of the transduction/transfection
considering that retroviruses have been used for this purpose for the systems require actively dividing cells.
last 30 years. Nevertheless, both types of integrative viral vectors are Because activation of T cells occurs physiologically through
considered potentially oncogenic and need to be carefully tested for their interaction with myeloid cells, such as dendritic cells, these
the absence of replication-competent particles and require extensive primary cells have been tested as a system for T cell activation in
patient follow-up when used for clinical applications. vitro.[27] However, the manufacture of these natural antigen
Instead of viral vectors, which require extensive and expensive presenting cells (APCs) is time-consuming, expensive, and has
biosafety testing, transposon/transposase systems, which are variable success from donor to donor, which hampers their use
usually delivered into T cells by electroporation with plasmids, as a reliable source for T cell activation. To overcome this
can be used. These systems have lower costs, simpler limitation, artificial APCs, such as the chronic myeloid leukemia
manufacturing procedures, and are potentially safer since cell line K562, have been used as feeder cells, allowing consistent
insertions have a nearly random distribution.[23] However, these T cell activation.[28] These artificial APCs can be modified to
systems have disadvantages, including lower transduction express costimulatory molecules (such as CD40, CD80, and
efficiency, which may require prolonged periods of CAR-T cell CD86) that further help with T cell stimulation. Additionally,
expansion that may potentially reduce their antitumor effect in artificial APCs can be further engineered to express the antigen
vivo. The efficacy of CAR-T cells generated using non-viral targeted by the CAR, leading to selective expansion of CARþ T
systems remains to be demonstrated, as there has been only a cells once the artificial receptor gets expressed (shown, e.g., with
small number of clinical trials using them, but modest a PSCA-specific CAR).[29] Most often, however, simpler alter-
antitumor activity was observed in a clinical trial using CD19 natives employing T-cell activating CD3 and CD28 monoclonal
CAR-T cells transfected with a Sleeping Beauty (a form of antibodies are used for T cell stimulation;[30] these antibodies
transposon/transposase) system.[24] coat plasticware or are attached to suspension beads present
Another option for T cell transduction is the electroporation of during T cell culture. Any of these methods leads to the
mRNA encoding the CAR into to their cytoplasm.[24] With this generation of T cells with high replicative capacity.
strategy, there is no integration of an exogenous gene into the T cells are then transduced or electroporated to introduce the
genome, mitigating concerns regarding genotoxicity. This CAR gene and expanded in culture for days to weeks (Figure 2).
system has already been tested in clinic[25] and CAR expression The primary goal of this step is to generate sufficient cells (up to
was detected on the T cell surface only for up to 1 week. Since 1011) to inject back into the patient to produce adequate

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on the intrinsic attributes of each patient’s circulating PBMCs.

However, recent studies have suggested that certain subsets of T
cells, such as naïve[36] and central memory[36] populations, may
possess functional advantages, such as longer-term persistence,
better proliferative capacity in vivo, and prolonged survival after
adoptive cell transfer compared to more differentiated effector T
cells.[37,38] Injection of less differentiated T cells has been
positively correlated with antitumor response in clinical trials.[39]
The reductionist approach of selecting a particular T cell
subset might, however, exclude populations with lower cytotoxic
effect but with complementary function. For instance, although
the ratio of CD4þ to CD8þ T cells at the beginning of culture
reflects that of peripheral blood, ex vivo expansion of T cells often
favors the CD8þ subset. Although a pure CD8þ population is
expected to have better cytotoxic activity, the use of 1:1 ratio of
CD4þ:CD8þ CAR-T cells has improved antitumor effect in
preclinical models and showed impressive activity in a group of
Figure 2. Schematic representation of the manufacture process of CAR-T
cells used in clinical trials. T cells are collected from the patient usually patients with acute lymphoblastic leukemia.[40] CD4þ T cells do
through leukapheresis (1) and then activated (2) and transduced with a not merely enhance CD8þ T cell function by release of cytokines,
retroviral vector (3). CAR-T cells are then expanded (4) to obtain sufficient but they can also efficiently promote tumor elimination through
numbers to infuse back into the patient a few days or weeks later (5). their direct role in tumor rejection, as recently demonstrated.[41]
Most CAR clinical trials conducted to date have not selected
antitumor effects. Although commonly used in research labs, for a particular subset of T cells. However, if adjustment of
conventional static cell culture systems (i.e., plates and flat CD4þ:CD8þ ratios or enrichment for naïve/central memory
flasks) are expensive, labor intensive, and difficult to scale up. populations ultimately demonstrate superior antitumor effects,
CAR-T cell production for clinical trials has thus relied mostly on manufacturing protocols may need to be altered to include extra
bioreactor culture systems, which are able to hold large volumes purification steps. Clinical grade magnetic sorting for these Tcell
of culture medium and have documented advantages (reviewed subsets has been developed[42] and can be easily adopted for the
elsewhere).[31] Systems such as WAVE BioreactorsTM (GE generation of CAR-T cells if required. These sorting procedures
Healthcare Bio-Sciences, USA) or the more recent G-RexTM may even be helpful for isolating bulk T cells from the rest of the
(Wilson Wolf, USA) have been used in several forms of ATCT PBMCs prior to starting expansion, as some populations of
and in clinical trials of CAR-T cells.[32] The former consist of monocytes (which are collected concomitantly during leukaphe-
single-use cell culture bags that are placed on top of a rocking resis) can inhibit CAR-T cell expansion,[43] a finding that may
base that maintains a constant temperature and enables gas explain failure of CAR-T cell production from some patients.
transfer and mixing; the latter use tall flasks that have a gas-
permeable membrane on their base that allows gas exchange
through convection, permitting maximum cell growth with 6. Scalability of Autologous CAR-T Cell
uninterrupted access to nutrients. Both systems allow fast
Therapy
expansion, with capacity for scaling up. With recent improve-
ments, CAR-T expansion requires usually 7–14 days until an To date, the cost and complexity associated with manufacturing
adequate number of cells is attained. restricts CAR-T cell production to specialized centers that have
Although Tcells expand well in vitro, a balance is also required the capacity to deliver the therapy to the small number of patients
between maximizing T cell numbers during expansion and required for early phase clinical trials. A jump from “proof-of-
preventing their differentiation and exhaustion, because the concept trials” to commercialization will require a paradigm
latter leads to T cell senescence and limited persistence in shift from the way conventional drugs and other biologic
vivo.[33] During expansion, T cells are exposed to a combination therapies are generated.
of proinflamatory cytokines that promote their growth but can Regardless of the specific CAR being used, CAR-T cell
also prevent their full differentiation, improving their persis- production needs to occur according to a set of rules that are
tence, and reactivity when injected back into patients.[34,35] defined under Good Manufacturing Practice (GMP) guidelines.
Different institutions use a combination of different cytokines Thus far, CAR-T cell generation in GMP facilities is performed
such as IL-2, IL-7 and IL-15, or IL-15 and IL-21, but whether any using protocols adapted from basic research, often in open
particular cocktail has advantages over the others is an area of systems that require constant manipulation by highly skilled
ongoing research. Nevertheless, the dual role of IL-2 in technicians. These requirements preclude production of several
expanding conventional and regulatory T cells[33] may be a CAR-T cell products in parallel without exponentially increasing
drawback to using it in this context. the number of technicians and space needed, associated costs,
To date, CAR-T cells used in most clinical trials have been and risk of failure and contamination. They also complicate the
expanded from a heterogeneous PBMC population without any design of automated systems due to the complex nature of the
form of selection before manufacture and administration, protocols, which entail many different steps performed over a
leading to a final product whose characteristics depend mostly short period of time. While some of the bioreactors described

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earlier allow fast expansion of T cells, CAR-T cell production must also be considered. Nevertheless, although an expensive
depends on complex and integrated steps, especially during therapeutic approach, its potential economic benefits for the
genetic modification, which are usually carried in independent, patient and society also need to be considered in any financial
uncoupled systems. Under this circumstances, personalized cell analyses.
therapies such as CAR-T cells cannot easily become widely
available. If CAR-T cell processing became more automated, cell
products could be produced for a greater number of patients 8. Toward Universal CAR-T Cells
more efficiently.
Conversion of these protocols, which rely on multiple Ideally, any commercial cellular therapy product should allow
manual manipulations during activation, transduction, feed- prefabrication, be collected from a healthy third party, be easy to
ing, expansion, washing, and harvesting, to fully sealed robotic store, and be quickly delivered as an off-the-shelf product.
procedures has proven difficult but will likely be required to Several solutions are already being tested to overcome the
reduce the risk of contamination and the overall costs limitations of using autologous T cells.
associated with labor, laboratory usage, and materials. Closed One approach is to insert the CAR into other types of cells that
and automated systems are now being developed, such as the can also be adoptively transferred. Natural Killer (NK), NK-T, and
CliniMACS ProdigyTM designed by Miltenyi Biotec, which γδ T cells collected from a healthy donor may be able to replace
combines a cell washer that can purify immune cells directly autologous conventional T cells for CAR-cell generation and be
from blood, a magnetic cell separation system, and a cell used in cancer patients without causing graft-versus-host disease
cultivation device that also allows viral transduction of T (GvHD). A potential advantage of NK cells over T cells is their
cells,[44] all of which may reduce the need for large sterile innate potential antitumor activity: even if antigen loss renders
rooms. Still one limitation of this type of all-in-one machines is tumor cells invisible to the CAR, CAR-NK cells will still express
that during one run of T-cell expansion, the system cannot be an array of receptors that may recognize aberrant expression of
used to produce CAR-T cells from another donor, and thus its TAA in cancer cells.[48–50] However, whether any of these other
scalability is directly dependent on the number of machines immune cells has similar antitumor response is still unknown
available to run in parallel. because they have never been tested with a CAR in a clinical trial.
On the other hand, patient-specific cell products can only be In addition, these cells may have some limitations as they can
used for one patient per batch, and thus one of the biggest still be recognized and eliminated by the host immune system;
limitations of current CAR-T cell production lies in its they do not seem to survive as long as T cells in vivo, at least in
personalized nature. Irrespective of the system used to produce part because of the lack of a memory population; and some of
CAR-T cells, large-scale production of a genetically engineered them (e.g., NK cells) may not be easily frozen without decreasing
autologous cell product poses a number of challenges regarding their antitumor activity.[51]
manufacture timing and cost. Scaling up the manufacturing The use of an immortalized cell line, which is easily
process of patient-specific cells requires the capacity to generate expanded and scalable, is a potential solution to prevent
multiple independent expansions of T cells in parallel. While the variability among batches. Thus far, there is no immortalized T-
conventional biopharmaceutical economic model follows the cell line available for clinical use, but a continuously growing
principle that costs are reduced by scaling up production, the NK cell line (NK-92) is available.[52] This solution would be
cost per batch of CAR-T cells (specific for each patient) cannot be limited to a few countries since, for example, the European
reduced by producing a larger batch. Thus, since it is regulatory agency did not approve their use. Nonetheless,
personalized for each individual, CAR-T cell manufacturing clinical trials using NK-92 cells are ongoing in China
may not have an economy of scale. Nonetheless, improvements (NCT02944162, NCT02892695). If these current clinical trials
in engineering and manufacturing technology, the use of closed show successful results and demonstrate safety, increased
systems, and automation of complex, labor-intensive steps attention to this technology is expected.
should improve scalability to some degree, which in turn should The generation of allogeneic “universal,” off-the-shelf T cells
reduce the costs.[45] is also under intense investigation. These platforms use genome
editing tools to inactivate the endogenous TCR, preventing
modified T cells from producing GvHD.[53,54] Disrupting the
TCR at the same time that the CAR is introduced allows the Tcell
7. Cost of CAR T Cell Technology
to be activated only through the CAR. Off-the-shelf CAR-T cells
Although it is too soon to assess real world amounts, it has been have shown efficacy in lymphoma xenograft models and in two
estimated that production of CAR-T cells for a single patient infant patients who were treated for B-cell leukemia with CD19
may reach up to $40 000.[46] Such costs are due to the need for CAR-T cells that had their endogenous TCRs ablated by TALEN
sophisticated manufacturing facilities, highly trained person- technology and were magnetically depleted of TCRþ cells (<1%
nel, expensive materials, and assurance of biosafety and quality. TCRþ T cells remaining).[55] Of note, GvHD was still observed in
The commercial costs of CAR-T cell therapy are, however, these patients, although mild and limited only to skin, in
expected to be much higher than just that of CAR-T cell contrast to the more widespread manifestations observed in
production,[47] likely around 10 times or more that amount. In typical GvHD; and there was evidence of alloreactivity in the
addition, when estimating the total cost of CAR-T cell therapy, marrow. In any case, this experience demonstrates that sorting of
other expenses, such as that of hospitalization and co- any particular subset may not be enough to totally prevent
administration of other agents or drugs with the CAR-T cells, potential complications from allogeneic T cell injection.

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Production of off-the-shelf CAR-T cells faces problems 9. Future Directions

similar to that of autologous CAR-T cells, since it requires
Although CARs allow targeting of tumor cells by T cells, most
similar expansion protocols, in addition to the extra steps of
cancers and their supporting stroma have evolved a multitude of
TCR knockout and selection of TCR-negative cells (Figure 3).
ways to evade recognition by immune effectors and thus prevent
Nonetheless, each batch could be used to treat more than one
tumor killing by T cells. Further T cell engineering may thus be
patient, thus decreasing dramatically the cost of production
required to extend the success observed in hematologic
per patient, which may push adoptive cell therapy toward an
malignancies to solid tumors, which so far have been much more
industrialization and standardization production model with
difficult to address. Examples of these additional genetic
consistent pharmaceutical release criteria from batch to batch.
modifications include forcing the expression of chemokine
Although the idea of universal CAR-T cells is attractive, their
receptors to increase trafficking and survival to specific tumor
safety and efficacy has not yet been proven, and further
sites such as the brain;[59] increasing cytokine production to
research is required before this approach could be
modulate the tumor microenvironment;[60,61] making T cells
implemented.
resistant to inhibitory signals;[62] or inserting artificial receptors
Finally, the search for a “universal” effector is also being done
that change how Tcells perceive inhibitory signals from the tumor
with the CAR itself. While most CARs currently being tested in
microenvironment.[63,64] Alternatively, targeting simultaneously
the clinic have been designed for a specific target according to
immunoinhibitory pathways, including, for example, combining
the scFv in their antigen binding domains, a new line of research
checkpoint blockade with CAR-T cells,[65] may provide additional
is developing a universal CAR that recognizes a soluble molecule
benefits. All these approaches are the focus of current research.
that acts as a bridge between the CAR itself and the target
molecule. This bridging molecule is recognized by the CAR after
it binds to the target molecule on the tumor cell surface, thus
directing the CAR-T cells to specific antigens.[56,57] This
10. Conclusions
technology would allow the generation of one T cell product Adoptive cell therapy has entered a new phase in which genetic
for all types of cancers. Since it would be possible to target transfer of tumor-specific receptors into T cells can convert them
multiple antigens simultaneously with different bridging into effective cancer killers. CARs allow antigen recognition
molecules, this approach may also reduce the risk of tumor without MHC restriction and thus have removed one of the
antigen escape variants. Clinical trials using this approach are in obstacles to more widespread application of T cell therapy. CAR-T
progress (NCT02776813).[58] cells are highly targeted, like monoclonal antibodies, but possess
the additional benefits of active trafficking to cancer sites, in vivo
expansion and long-term persistence. Moreover, further genetic
modifications can serve as countermeasures to tumor immune
evasion strategies. Optimized CAR design, better understanding
of the processes leading to T cell activation and memory, and
improved gene transfer methods have allowed this strategy to be
successfully brought to the clinic. In addition, technical improve-
ments in cell processing, such as automation and better culture
systems, will likely lead to simpler and faster CAR-T cell
production, hopefully driving down its costs. Challenges regarding
scalability and changes in manufacturing models to allow mass
production remain hurdles to the widespread use of CAR therapy.
Nonetheless, the striking results obtained with CD19-specific
CAR-T cells support the idea that this therapy will soon join the
mainstream of oncologic treatment, a noteworthy instance of a
concept taken from the research bench to the assembly line.

Abbreviations
ATCT, adoptive T-cell therapy; CAR, chimeric antigen receptor; GMP, good
manufacturing practices; GvHD, graft-versus-host disease; HLA, human
leucocyte antigen; MHC, major histocompatibility complex; PBMC,
peripheral blood mononuclear cells; scFv, single-chain fragment variable;
TAA, tumor associated antigen; TCR, T cell receptor; TILs, tumor
infiltrating lymphocytes.

Figure 3. Workflow of CAR-T cell manufacturing. Starting with blood

collection, T cells undergo a complex process evolving multiple sequential Acknowledgements
steps until infusion into the patient. Each step is performed in GMP
conditions with several alternatives from institution to institution. Steps The authors thank Catherine Gillespie for editing the manuscript. Diogo
in blue are usually required for generation of “off-the-shelf” CAR T cells. Gomes-Silva acknowledges FundaSc ~ao para a Ci^encia e Tecnologia (FCT,

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