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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5866_Библиотеки_им_академика_М_И_Перельмана
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CARs are commonly formed by a specific conferring single-chain variable fragment
(scFv) derived from a B cell receptor, a CD3z domain derived from a TCR, and one
or more intracellular costimulat ory domains. Importantly, over years several CAR
designs have been proposed to incre ase the safety, specificity, and efficacy [1].Sofar,
five generati ons of CARs have been developed with each generation including a
new intracellular domain to enhance their desirable properties in the effort to eliminate
cancerous cells (Fig. 1).
Following initiatives at the Massachusetts Institute of Technology, the first generation
of CAR was developed in 1993 at the Weizmann Institute of Science in Israel. This initial
model consisted of only the transmembrane domain CD3z [2]. A major problem with
this early CAR was that it did not persist in the long-term in vivo studies which resulted
in poor clinical efficacy [3]. It was not until 5 years later that a scientific team working at
the Memorial Sloan Kettering Cancer Center in New York suggested that the addition of
a costimulation of CD28 increased the survival and proliferation of CAR-T cells [4]. The
second generation of CAR-T cells saw the introduction of a costimulatory domain such
as CD28 or 4-1BB. As a result, these cells began to show desirable properties such as
enhanced proliferation and a better survival profile [5]. In particular, 4-1BB-CAR-T
cells seemed to have a longer persistence compared with CD28-CAR-T cells, but the
early loss of effector function (exhaustion) due to prolonged stimulation by antigen might
limit the antitumor efficacy [6]. The CD28-CAR-T cells cause constitutive stimulation,
proliferation, and growth of the CAR-T cell [7].
3Engineering solutions to design CAR-T cells
Fig. 1 Diagram showing different CAR generations . The fi rst generation of CAR has only one intracellular domain, while the second generation has a co-stimulatory molecule (either CD-28 or 4-1BB).
In the third generation, a second co-stimu latory molecule is added and the fourth generation
is designed with a cytokine inducer to locally release an inducible transgenic cytokine (for example
IL-12). In the fifth generation an IL-2 receptor β-chain (IL2RB) is included. (Created with BioRender.
com.)
4 Irene Uboldi et al.
To increase the potency with stronger cytokine production and killing ability, a
third-generation was designed. This third generation of CAR contained a combination
of multiple signaling domains such as CD3z-CD28-OX40, CD3z-CD28-41BB, or
CD28-41BB [7]. However, the third generation CARs did not show better outcomes
than the second generation [8,9]. Furthermore, a relapse in the tumor was observed
in patients after an initial reduction of the tumor by CAR-T cells therapy. The reason
for this was the loss of the target antigen in cancer cells [10]. To address this issue, a
fourth-generation of CAR was created. This generation was designed to locally release
an inducible transgenic cytokine, for example, IL-12. It is well known that IL-12
enhances T cell activation, recruits and activates innate immune cells to kill antigennegative cancer cells [10]. This localized production is controlled by a nuclear factor
of activated T cell (NFAT) responsive promotor [2]. The fourth-generation of
CAR-T cells with the transgenic payload is also called T cells Redirected for antigenUnrestricted Cytokine-initiated Killing (TRUCK) T cells [10]. The local production
of IL-12 instead of systemic administration is of great importance. In fact, in preclinical
models, infusion using IL-12 has shown great potential as an effective antitumor agent,
but owing to its severe clinical toxicity, early clinical trials were deemed unsuccessful.
The fourth-generation CAR-T cells addressed this issue by circumventing the need
for systemic infusion, but rather provided an engineered solution to produce IL-12
locally at the tumor site. The expectation was that this would result in low-serum
concentration of IL-12 and therefore lower toxicity. Koneru et al. have shown the
enhanced antitumor efficacy of IL-12 secreting CAR-T cells in a mice model with
human ovarian cancer xenograft [11].
The fourth-generation CAR-T cells did not show therapeutic efficacy for several
types of malignancy, and therefore, a fifth-generation was very much in need [12].
The fifth-generation of CAR is currently under development. Here an IL-2 receptor
β-chain (IL2RB) domain was included [2]. The IL2RB is incorporated between the
costimulatory domain CD28 and TR signaling CD247 and was particularly adopted
to induce cytokine signaling. Besides, it was shown that these CAR-T cells promote cell
proliferation and prevent terminal differentiation. Besides, enhanced persistence and
therapeutic efficacy in the leukemia model were demonstrated [13].
The choice of the costimulatory domain is of great importance since it influences the
outcomes of the therapy. Costimulatory domain4-1BB is generally believed to differentiate
into central memory T-cells, while the CD28 confers to T cells a greater proliferation and
persistence [14].
The most challenging part in the engineering of CAR-T cells is the selection of the
target antigen. Ideally, the antigen should be expressed only on cancer cells and not on
healthy tissue to prevent any “on-target off-tumor” toxicity. But, to-date specificity
toward malignant cells has not been achieved even in the already approved CAR-T cells
therapies [15].
1.2 Current approved CAR-T cell therapies
To date, three CAR-T cell therapies have been approved by the United States Food and
Drug Administration (FDA). Namely Tisagencleucel, Axicabtagene ciloleucel and
lisocabtagene maraleucel, commercially known as Kymriah, Yescarta and Breyanzi,
respectively. The latter being the newest in the group and was approved by the FDA
on the 05th February 2021. These former two therapies have also been approved by
the European Medicines Agency (EMA).
Novartis currently holds the marketing authorization for Kymriah. It was approved
by the FDA on August 30th, 2017 for the treatment of large B-cell lymphomas in adults
and of B-cell precursor acute lymphoblastic leukemia (ALL) in patients under the age of
25. Kymriah was designed with a second-generation CAR containing the costimulatory
domain 4-1BB and it is designed to target CD19 antigen in tumor cells [14].
The marketing authorization for Yescarta on the other hand is held by Kite Pharma. It
was approved by the FDA on October 17th, 2017 for the treatment of B-cell lymphoma
in adults and patients with relapsed or refractory disease with a history of two or more
lines of systemic therapy [14]. Yescarta also adopted the second generation CAR but used
CD28 as a costimulatory molecule [14].
Currently, both therapies are under evaluation for other hematological neoplasms and
other different solid tumors.
5Engineering solutions to design CAR-T cells
1.3 Pharmaceutical manufacturing process of CAR-T cell
The manufacturing process of cell and gene therapy products often is very complex. The
reason for this is often attributed to the complexity in the steps required to process and
manipulate the cells to achieve desired specifications. Further, the stringent regulatory
requirement to ensure the safety of the product adds to the additional quality requirements in their GMP production.
Essentially, the production process for an autologous CAR-T cell begins with the
collection of cells from the patient via apheresis (Fig. 2). Apheresis is a simple yet smart
technological solution where blood from the patient is separated into specific components. The desired component is then collected. In this case, the Lymphocytes are of
interest. Once enough lymphocytic cells are collected, the remaining blood is pumped
back to the patient. These collected cells are then immediately cryopreserved or stored
fresh at refrigerated conditions. Assisted with appropriate packaging and temperaturecontrolled shippers, these extracted patient materials are then sent to a GMP facility
for further processing.
Under a cleanroom environment, the T cells are isolated and enriched. Various
methods such as the gradient density method and magnetic-bead-labeled antibodies
are employed for this purpose [16]. Ficoll-Paque, Magnisort, and Easy Sep are examples
of the ready-to-use kits available in the market that could be easily used. The recovered
6 Irene Uboldi et al.
Fig. 2 Schematic representation for the manufacturing process of CAR-T cells. Cells are collected from
patients via apheresis and subsequently lymphocytes are isolated from the other blood components.
Thereafter, they are activated and transfected with the CAR gene. The generated CAR-T cells are
expanded and cryopreserved. CAR-T cells are ready to be ship back to the hospital and be infused into
the patient. (Created with BioRender.com.)
cells are then, cultivated activated, transduced, expressed, and washed. Invitrogen’s CTS
Dynabeads, Miltenyi MACS ExpAct Treg, or TransAct beads are some of the readily
available kits that could be used for T cell activation. Equipment, such as the CliniMACS
Plus and Prodigy systems, is used for the enrichment of specific T cells, such as those with
+
CD4
, CD8+, or CD25. Further purification is achieved using devices, such as Fresenius
Kabi LOVO, are used. The final processed cells are then temperature preserved and
shipped to the clinic where the same patient from whom the cells were collected are
infused (autologous). Currently approved CAR-T cell therapies are autologous and
patient-specific [17].
The manufacturing process in which autologous CAR-T cells are produced impedes
its wide application. More often than not there is a variance inconsistency in the end
product. This is attributed to the variance in the starting material. The adoption of allogeneic T cells for the preparation of universal CAR-T cells holds great promise in its
immediate availability for use and its ability to reach a large number of patients and also
potential reduction of the costs of treatment. The universal CAR-T cells can be if successful can offer an “off-the-shelf” product solution that could be simultaneously be
administered to multiple patients [18]. Various studies are currently focusing on creating
a universal CAR to construct a versatile T cell, by expanding the antigen recognition.
This can be achieved by splitting the antigen-targeting domain and the T cell signaling
unit [18].
Besides, split universal and programmable (SUPRA) CARs are under development to
increase the flexibility and controllability of CARs. SUPRA CAR also consists of two
components receptor, one is a universal receptor with a leucine zipper adaptor (zipCAR)
and the other is a molecule targeting specific antigens [18].
One of the challenging steps in CAR-T cell manufacturing is the extraction of T cells
from the other blood components after apheresis. In fact, the initial quality of the collected T lymphocytes highly influences the time required to generate CAR-T cells
and the quality of the finished product. Currently, there is no standard method of purification. The two most known methods are magnetically activated cell sorting (MACS)
and fluorescence-activated cell sorting (FACS). MACS is a technology that uses magnetic
beads conjugated to specific antibodies to isolate cells. FACS instead labels cells using
fluorescence-tagged biochemical antibodies. The MACS technology requires less expensive equipment [19]. Notably, during MACS sorts there is a loss of cells between 7%
and 9%, rather than FACS where the percentage grows up to 70%. Comparing the
processing time of the two technologies, MACS is faster than FACS also due to its ability
to process multiple samples in parallel. For both methods, the cell viability remains high
(more than 83%) [20].
Currently, there are no uniform technical standards for CAR-T cell products. The
manufacturing process of CAR-T cells should meet the current good manufacturing
practice (cGMPs). Since CAR-T cell is a “living drug,” it requires a complex preparation
process and thus a complex in-process quality control. The general principles of quality
control are control of production material, in-process control, and release testing. Validation of the production process, defined stability study protocol are prerequisites to
manufacturing. The control of raw material that goes into production, consists of identification and quality testing of the substances or materials used in the preparation of
CAR-T cells. In-process control and testing are the controls performed during all process
are necessary to ensure the quality of the produced cells. These tests are important to
achieve repeatability and batch-to-batch consistency. Release testing is conducted to
ensure the identity, purity, safety, and effectiveness of CAR-T cells. Validation of the
production process is required to ensure the consistency of the process and the products.
The stability study is needed to ensure that the product is still usable during the specified
shelf life of the finished product. For example, shelf life can be compromised by the presence of bacteria and mycoplasma contamination in the product [21].
7Engineering solutions to design CAR-T cells
8 Irene Uboldi et al.
2. Pharmacological aspects of CAR-T cells
2.1 Mechanism of action of CAR-T cell therapy
CAR-T cells, when infused into the patient, migrate to the tumor and specifically
recognize tumor cells using their specialized extracellular receptors. Once attached,
the cascade of intracellular signaling pathway begins, resulting in the elimination of
the tumor cells. In addition to directly affecting tumor cells, CAR-T cells, proliferate
and release a variety of inflammatory cytokines via CAR-mediated signaling [22].
2.2 Efficacy of current CAR-T therapies (Kymriah and Yescarta)
Novartis Pharmaceuticals is currently conducting an ELIANA clinical trial for the testing
of Kymriah in patients with B-cell ALL. The trial started in 2015 and is expected to end in
2022. The patients enrolled are pediatric and young adult patients who had a primary
refractory, chemorefractory, or relapsed after, or were not eligible for allogeneic stem
cell therapy. The overall remission rate reported within 3 months after the infusion
was 82%, which includes complete remission and complete remission with incomplete
blood count recovery. Furthermore, 75% of responders were in remission at 6 months
after infusion and at 12 months the responders still in remission were 64%. The overall
survival at 6 and 12 months was 90% and 76%, respectively [23,24].
Importantly, the quality of life of the patients infused with Kymriah improved within
3 months. This improvement only doubled within the next 12 months. Following
3 years of the treatment, the number of patients still in remission reduced to 45%. This
is mostly due to the loss of CD19 antigen or epitope in tumor cells. It was also shown that
antigen escape is one of the mechanisms by which tumor cells resist CAR-T cell therapy
and that about 27% of relapses are due to antigen loss [25,26].
While the data from a multicentered ZUMA-1 trial where Yescarta was tested,
showed that at least 39% of the patients remained in remission for over 2 years
(clinicaltrial.gov identifier: NCT02348216). This is exciting as evidence suggests that
patients that remained in remission for over 2 years have a greater chance of the lymphoma never relapsing.
2.3 Side effects of current therapies (Kymriah and Yescarta)
CAR-T cells are designed to kill the target cancer cells and also ensuring an increased
phenotypic population in vivo. These mechanisms are mediated by the release of antitumor cytokines. The downside of this therapy is that the recruitment of more T cells
leads to a large release of cytokines with the risk of reaching a toxic level. This is presented
in the patient in the form of cytokine release syndrome (CRS). CRS in severe cases can
result in patient death [26]. The severity of CRS is graded on a scale from 1 to 4 where
grade 1 means mild reaction with fever or mild organ toxicity, while grade 4 means lifethreatening consequences as hypotension requiring high-dose vasopressors and hypoxia
requiring mechanical ventilation [27].
Another serious side effect of CAR-T cell therapy that could lead to death is neurotoxicity [26]. But one of the notable and potentially serious side effects is the “on-target
off-tumor” toxicity where CAR-T cells may react against normal tissue that expresses the
targeted antigen [26].
2.4 Challenges in CAR-T cell therapy
CAR-T cell therapy has so far shown the most promising results in patients with ALL.
However, yet only some of the patients achieved a lasting response, while the occurrence
of possible relapse or the lack of response remain major clinical challenges. The reasons
for the limited effects are the lack of persistence of CAR-T cells and down regulation of
target antigen [28].
Tumor antigens are usually expressed heterogeneously and are often downregulated
in cancer cells as an inherent mechanism of avoiding detection by immune cells. Both
these reasons could lead to a clonal escape from CAR-T cell therapy. Furthermore, solid
tumors present additional challenges. In addition to the lack of specific antigens to target,
homing of CAR-T cells in the solid tumor tissue poses a great challenge. This is due to
the presence of extensive extracellular matrix (ECM) and high interstitial fluid pressure
within the tumor microenvironment (TME) [29].
Also, tumor antigens expressed in solid tumors targeted by CARs also express in healthy
tissues and the lack of specific tumor antigen raises the risk of on-target off-tumor toxicity.
Evidently, there is a reported case of a patient with metastatic colon cancer treated with
CAR-T cells targeting human epidermal growth factor receptor (HER2), a tyrosine
kinases receptor overexpressed in colorectal cancer. The patient died after 5 days of the
infusion and the death was attributed to the attack of CAR-T cells to the epithelial cells
of the lung that also expressed low levels of HER2 [9,30]. Similarly, on-target off-tumor
toxicity was reported in a preclinical model of a human neuroblastoma xenograft in vivo.
The tumor-associated antigen GD2 highly expressed in neuroblastoma was targeted by
high-affinity antiGD2 CAR-T cells to treat the neuroblastoma. They found that low levels
of GD2 in the brain resulted in fatal encephalitis [31,32].
Even after the successful delivery of the CAR-T cells to the vicinity of the tumor,
there are multiple challenges to overcome. In particular, the altered condition of the
TME such as low pH and the limited amount of oxygen and nutrients. Furthermore,
the constant exposure to the target antigen with a lack of sufficient stimulation can lead
to CAR-T cell hypofunction [33].
9Engineering solutions to design CAR-T cells
10 Irene Uboldi et al.
3. What can be engineered in CAR T-cells?
3.1 Engineering CAR-T cells to secrete a product
CAR-T cells can be engineered to secrete various types of products such as cytokines,
matrix-degrading enzymes, bispecific T cell engagers (BiTEs), and antibodies (Fig. 3,
Table 1).
3.1.1 CAR-T cells secreting cytokines
Signals play a central role in the activation and proliferation of T cells. For an optimal
stimulation of T cells, there is the need for a flawless orchestration of these signals. Signal
1 is produced by the engagement of the T cell receptor, signal 2 is produced by the
costimulatory domain which confers the T cell its effector function, induces proliferation, and confirms persistence. Signal 3 is produced when the cytokines are engaged
[12,48]. The importance of signal 3 for CAR-T cell function is highlighted in several
studies in which the cytokine expression improved persistence and antitumor activity
[34,49]. Various approaches have been tested to augment the presence of cytokines.
These strategies include systemic administration of cytokines and CAR-T cells that constitutively express various cytokines. However, both solutions have been shown to cause
serious adverse effects such as cytokine release syndrome (CRS), neurotoxicity, and
potentially worsened cancer growth [12]. To prevent these side effects, there is a growing
need to engineer CAR-T cells that locally deliver cytokines.
Several researchers have developed engineered CAR-T cells to secrete IL-12 [10,34,35].
These were shown to enhance the antitumor activity by increasing the survival and the
Fig. 3 CAR-T cells engineered to secret products. CAR-T cells can be engineered to secrete various
products such as matrix degrading enzymes, bispecific T cell engagers (BiTEs), antibodies and oncolytic
viruses. (Created with BioRender.com.)
Table 1 CAR-T cells to produce a product.
Name of the CAR T-cell Product secreted For what it is used References
Armored CAR T cells
to produce cytokines
IL-12 [10,34,35]
IL-15 [36]
IL-18 [37]
IL-21 Support to CAR T cells
IL-27 with signal 3
IL-23
Il-36γ
Co-expression of IL-5, IL-21 [38]
Co-expression of IL-7, IL-15, IL-21
Delivery of chemokines CCL19 Recruiting T cells
Co-expression of CCL5, CXCL9 [39]
Secretion of extracellular Heparinase enzymes To degrade the ECM [40]
matrix-degrading enzymes
Secretion of BiTE CD-19-targeted BiTE Allow a close interaction between
[41–43]
T cells and cancer cells
Secretion of antibodies
Anti-PD-L1 To prevent T cells [44]
Anti-PD-1 exhaustion [45]
CTLA-4 [46]
Delivery of TGFβ inhibitors TGFβ
Deposit of viruses in TME Oncolytic viruses Cause specific [42,47]
Soluble herpesvirus Tumor cells lysis
12 Irene Uboldi et al.
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persistence of CAR-T cells [11]. Furthermore, the cytokine expression can be delicately controlled to precise release within the TME. This is achieved by introducing the cytokine gene
under the influence of a nuclear factor of activated T cells (NFAT). This inducible promoter is
expressed exclusively when the T cell is activated. Studies have shown a positive outcome
with a higher antitumor activity of CAR-T cells both in vivo and in vitro [10,34,35].
To further understand and possibly increase the antitumor property, various scientific
groups have produced CAR-T cells that produce IL-15 and IL-18 [36,37]. Another cyto-
kine engineered to be secreted by CAR-T cells is IL-21. This is now being tested in clinical
trials (NCT04093648). Further efforts are in progress to design CAR-T cells that can
secrete multiple cytokines at the desired time and location in a time-controlled mode.
Preliminary studies using CAR-T cells coexpressing IL-15 and IL-21 on the hepatocellular carcinoma murine model showed appreciable antitumor activity [38]. Batra
et al. designed a combinatorial secretion of IL-7 and CCL19 by CAR-T cells. This combination showed complete remission in the mouse model. This study also identified the
presence of memory response against the tumor [50]. Other promising cytokines need
further in-depth investigation. For example, IL-27 is shown to increase the antitumor
activity of T cells and induce a precursor of the T cell memory phenotype which is associated with the ability of self-renewal [51]. Another study by Dangaj et al. showed that
coexpression of the chemokines CXCL9 and CCL5 was associated with greater infiltration of T cells into solid tumors [39].
3.1.2 CAR-T cells secreting extracellular matrix-degrading enzymes
CAR-T cells showed less astonishing results in solid tumors when compared with lymphoid
malignancies [3,52]. The limiting efficacy could be partially caused by functional changes in
CAR-T cells after the ex vivo manipulation to generate them. In fact, in vitro cultured lymphocytes exhibited an inferior capacity to degrade extracellular matrix (ECM) components
in contrast with freshly isolated T lymphocytes. The inferior degradation capacity maybe
account for cultured CAR-Tcells as well and thus compromise their extravasation and penetration into the solid tumor [40]. When the culturedT cells wereanalyzed,it was found that
they lack expression of the enzyme heparinase (HPSE).Essentially, HPSE degrades heparan
sulfate proteoglycans, which is the major component of the ECM.
To address this problem, engineering CAR-T cells to secrete HPSE has been considered. Studies carried out in preclinical models using this approach have shown enhanced
T cells infiltration into neuroblastoma and thereby an increased antitumor activity [40].
3.1.3 CAR-T cells secreting BiTEs
Bispecific T cell engagers (BiTEs) bring together two single-chain variable fragments
(scFvs) from different antibodies. This cleverly designed protein pair offers two arms,
one that is specific for a cell surface molecule on the T cell, for example, CD3, while
the other is specific to the cancer cell. Both of these are covalently bonded by small linker
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