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Cancer stem cells 363
Adipocytes
Endothelial
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patients with constitutive PI3K have a favorable prognosis,
which is proposed to be due to PI3K driving LSCs into the S
phase of the cell cycle, thus making them more sensitive to
chemotherapy.
Targeting LSC dormancy
Chemoresistance is often attributed to CSCs, which due to
their largely quiescent nature can evade the effects of chemotherapy agents that preferentially target actively cycling cells.
Breaking the dormancy of LSCs therefore represents a
rational approach to eliminate the LSC population. In murine
models, AML cells residing in the endosteal region, where
quiescent, chemo- resistant LSCs preferentially home to, have
been shown to enter the cell cycle upon treatment with the
mobilizing agent granulocyte colony- stimulating factor
(GCSF). Furthermore, addition of GCSF to cytarabine
resulted in enhanced cell death and elimination of LSCs, as
assayed by limiting dilution analysis. These findings are supported by clinical data from newly diagnosed patients with
AML who received GCSF in conjunction with induction
chemotherapy, showing improved outcomes in terms of
disease- free survival, although this benefit did not extend to
those with poor- risk disease, and no overall survival advantage
was noted. From a mechanistic point of view, it is unclear
whether the enhanced chemosensitivity observed with GCSF
relates solely to its effect on cell cycle progression, or whether
there are other mechanisms involved, such as displacement
of LSCs from the niche and potential synergistic interaction
with chemotherapy.
The use of arsenic trioxide, which is licensed for the treatment of PML/RARa acute promyelocytic leukemia, has also
been suggested as an alternative intervention to enhance the
cycling of LSCs and, consequently, their elimination. In a
CML mouse model, loss of PML led to LSC exhaustion and
diminished repopulating ability. Furthermore, PML inhibition with arsenic trioxide in both murine and human CMLLSC resulted in cell cycle induction, which was more
pronounced compared to normal HSC, and was associated
with enhanced chemosensitivity.
Targeting the CSC niche
The bone marrow niche represents a complex and dynamic
microenvironment that provides a host of signaling cues that
are crucial for determining the fate decision of normal HSCs.
As with HSCs, niche- derived signals are vital for maintaining
LSCs (see Figure24.2). Evidence from several murine models
Mesenchymal
Sympathetic neurons
Osteoclast
stroma cells
?
?
cells
Bone
Osteoblast
?
LSC
?
Blood vessel
?
Endosteal niche
Figure24.2 Schematic representation of the interplay between the niche and leukemic cells. The bone marrow (BM) niche consists of
several cellular components including mesenchymal stroma cells, osteoblasts, endothelial cells, adipocytes, and sympathetic neurons. The
leukemia/microenvironment interaction is a dynamic, bidirectional process whereby leukemic cells receive vital cues from the niche that influences
their behavior. Conversely, leukemic cells hijack and remodel the BM niche into a malignant environment that is more permissive to the disease at
the expense of normal hematopoiesis. LSC, leukemic stem cells.
Vascular niche
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even indicates that alterations in components of the bone
marrow microenvironment may be sufficient to disrupt normal hematopoiesis and induce neoplastic transformation. In
an elegant study by the Scadden group, selective deletion of
the miRNA processing endonuclease Dicer 1in osteoprogenitors, resulting in impaired osteoblastic differentiation, induced
profound hematopoietic defects mimicking myelodysplasia,
with subsequent transformation to myeloid sarcoma in some
mice. Intriguingly, the same phenotype was observed when
wild- type hematopoietic cells were transplanted into mice
with mutant niche, while the effect was reversed upon transplantation of hematopoietic cells from mutant mice into a
wild- type environment. This lends further support to the
concept of niche- driven oncogenesis, whereby microenvironmental aberrations constitute the primary pathogenic event
that drives tumorigenesis, which consequently facilitates the
acquisition of oncogenic hits in the hematopoietic compartment. Similarly, constitutive activation of β- catenin in murine
osteoblasts has been shown to alter the differentiation potential of lymphoid and myeloid progenitors, leading to a differentiation block and the emergence of AML through
upregulation of the Notch- ligand Jagged 1in osteoblasts and
subsequent increase of Notch signaling in leukemia- initiating
HSCs. However, it remains to be determined whether this
mechanism of niche- driven oncogenesis occurs in human
hematological malignancies.
The relationship between the niche and leukemic cells is
clearly not a one- way street and, just as the niche can dictate
tumor behavior, so too can leukemic cells reshape and
manipulate their microenvironment in their favor at the
expense of normal hematopoiesis. For example, in a mouse
model of BCR- ABL CML, reduced expression of CXCL12was
observed in the bone marrow stroma cells secondary to
secreted factors by leukemic cells, leading to reduced engraftment of normal HSCs, but not of BCR- ABL- positive HSCs.
Similarly, in a murine model of myeloproliferative neoplasm
(MPN), neoplastic myeloid cells were shown to remodel the
endosteal niche by promoting the differentiation of mesenchymal stroma cells toward the osteoblastic lineage. Notably,
the expanded osteoblasts were functionally altered, with
compromised ability to support normal HSCs but not LSCs.
AML-
induced alterations in endothelial cells resulting in
increased vascular permeability and sympathetic neuropathy
of the niche in MPN are among other reported examples of
neoplasia- induced remodeling of the bone marrow niche.
Given the important role of the niche in sustaining tumor
development, targeting the niche has been explored as a
potential therapeutic strategy. One approach has been to disrupt homing of LSCs by targeting the CXCL12/CXC4 axis in
leukemia. Pretreatment of AML cells with CXCR4 antibodies has been shown to impair their homing into the bone
marrow and spleen of transplanted mice. Conversely, mice
already engrafted with AML and treated with CXCR4neutralizing antibodies had a marked reduction in their
AML burden, but no significant difference in the level of
engraftment was observed in mice engrafted with cord blood
mononuclear cells, indicating that AML cells are more
CXCR4 dependent. Moreover, using a different small molecule
CXCL12/CXCR4inhibitor, CXCR4inhibition abrogated the
protection conferred by the stroma, resulting in increased
sensitivity to chemotherapy. Of note is that higher expression
of CXCR4 has been reported in patients with AML, and
CXCR4 expression level appeared to be an independent
prognostic indicator across a heterogeneous group of AML
patients with varying mutational backgrounds. Given the
role of CXCR4in homing, it is not unreasonable to speculate
that the negative impact on outcome may, at least in part, be
due to enhanced CXCR4-
mediated retention of LSCs.
Disrupting the LSC–niche interaction can also be achieved
by targeting the integrin very late antigen 4 (VLA4), which is
known for its important role in homing and retention of
HSCs in the niche. VLA- 4 binds to the fibronectin component of the extracellular matrix as well as the vascular cell
adhesion molecular- 1 (VCAM- 1), expressed by bone marrow stromal and endothelial cells, and inhibiting the VLA- 4/
VCAM- 1interaction induces mobilization of hematopoietic/
stem progenitor cells. In AML, interaction between VLA- 4
on leukemic cells and fibronectin has been shown to enhance
their survival and reduce their chemosensitivity through
activation of PI- 3k/AKT/BCL2 signaling. Combination therapy with the anti- VLA4monoclonal antibody Natalizumab
(NZM) and cytarabine resulted in increased AML apoptosis,
and mice treated with single- agent NZM had a significant
reduction in their AML burden.
Alternative therapeutic approaches include targeting the
adhesion molecule CD44. A monoclonal antibody directed
against CD44 resulted in a significant reduction of leukemic
burden in mice transplanted with AML. This effect was
mediated not only by impeding LSC homing, but also by
altering the fate of LSCs. A major drawback, however, is that
CD44 is also expressed in normal HSCs, and ligation of
CD44has been shown to affect the migratory behavior of
normal HSCs.
Targeting epigenetic modifiers
The use of epigenetic modulating therapies as a means to target CSCs in hematological malignancies has been mainly
explored in AML, in which mutations in epigenetic modifiers
are a frequent event, particularly in MLL- rearranged AML,
which is primarily driven by epigenetic dysregulation.
The DNA methyltransferase inhibitors, azacytidine and
decitabine, are currently the only two approved epigenetic
targeted therapies in AML. The effect of azacitidine on AML
LSCs has been investigated by Craddock and colleagues,
who found a substantial reduction in the LSC pool size,
although it was never eradicated, even in patients who
achieved complete morphological remission.
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Cancer stem cells 365
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Histone methyltransferase inhibitors are another class of
epigenetic therapies that have shown demonstrable effect
against LSCs. Key among these is 3- Deazaneplanocin A
(DZNep), which targets several components of the Polycomb
repressor complex 2, including EZH2. In a study by Zhou
and colleagues, DZNep demonstrated a significant antileukemic effect, including in LSC- enriched populations,
with no observed impact on normal HSCs in colony- forming
assays. However, more recently, loss of EZH2 has been
reported to correlate with poor prognosis and chemoresistance in AML, and treatment of AML cells with DZNeP was
found to induce resistance to cytarabine. In contrast to AML,
overexpression of EZH2has been reported in CML LSCs,
and inactivation of EZH2, in a conditional mouse model and
by clustered regularly interspaced short palindromic repeats
(CRISPR)/Cas9- mediated gene editing, has been shown to
block disease initiation and impair CML LSC function.
Similar findings were reported by Scott etal., who showed a
synergistic impact on the CML LSC compartment when
EZH2 inhibitors were used in conjunction with tyrosine
kinase inhibitors.
In addition to EZH2, the histone 3 lysine 79 (H3K79)
methyltransferase Dot1l has emerged as a critical regulator
of LSC activity. In MLL- AF9- induced leukemia, Dot1l has
been shown to play an important role in promoting LSC selfrenewal, and DOT1l inhibitors are currently being investigated in a number of clinical trials. Other histone modifiers
with the potential to modulate LSC activity in AML include
the histone demethylases KDM2b/JHDM1b and LSD1/
KMD1A.
Furthermore, anti- leukemic effect against AML LSC has
been shown with the histone deacetylase inhibitor AR- 42,
which causes inactivation of NF- κB, as well as inhibitors
against the chromatin reader Bromodomain- containing protein 4 (BRD4).
Other CSC- targeted therapies
LSCs can be distinguished from normal HSCs by virtue of
their distinct metabolic traits. In a study of chronic- phase
CML, the stem cell- enriched population exhibited an
increase in oxidative metabolism compared to normal HSCs,
and was more susceptible to oxidative phosphorylation inhibition when treated with combination of a tyrosine kinase
inhibitor and tigecycline, an antibiotic that inhibits mitochondrial protein translation. Reliance on oxidative phosphorylation rather than glycolysis has also been reported in
AML, where LSCs have been shown to have lower levels of
endogenous reactive oxygen species (ROS). Furthermore,
BCL- 2 was upregulated in ROS- low LSCs, which when
inhibited resulted in elimination of LSC both in vitro and
in vivo. Similarly, in AML with isocitrate dehydrogenase
(IDH) mutations, an RNA interference screen revealed synthetic lethality between IDH1 and BCL- 2. Treatment with
BCL- 2inhibitor- induced apoptosis in AML cells and affected
the survival of LSCs in xenotransplants through a mechanism involving 2- hydroxybutarate- mediated inhibition of
cytochrome c oxidase activity in the mitochondrial electron
transport chain.
The role of hypoxia in maintaining normal HSC functions, especially self- renewal, is well recognized. Similarly,
hypoxia is important for the maintenance of LSCs. Wang
et al. reported that in patients with AML, HIF1a and its
target GLUT1were selectively overexpressed in the LSC
fraction. Furthermore, inhibition of HIF- 1α by echinomycin induced apoptosis, which was more pronounced in the
LSC fraction compared to the AML bulk population. The
selective elimination of the LSC population by HIF inhibition was also demonstrated invivo by serial transplantation experiments, where mice injected with residual
leukemic cells from echinomycin- treated mice failed to
develop leukemia, indicating an absence of LSCs in the
residual leukemic population. Similar effects of HIF- 1α
inhibition have been reported in CML LSCs. It is worthy of
note, however, that the inhibition of HIF by echinomycin
is not restricted to HIF- 1α, and that the effect of echinomycin may, at least in part, be mediated by suppression of
HIF- 2α, which has been demonstrated to protect both normal HSC and AML cells from endoplasmic stress- induced
apoptosis.
Conclusion
The mainstay treatment in patients with hematological
malignancies remains traditional therapies that target the
tumor bulk but leave CSCs largely untouched, potentially
contributing to chemoresistance and disease relapse. In
recent years, tremendous advances have been made in our
understanding of CSC biology, which paved the way for the
development of a number of therapeutic agents that target
CSCs. While promising, several considerations should be
taken into account before CSCincorporated into routine clinical practice.
The timing of administration of a CSC- directed therapy is
likely to influence its efficacy. Ideally, a CSC- targeted therapy
should be given before standard chemotherapy when CSCs
are still in a naïve state, as the selective pressure exerted by
chemotherapy can inadvertently lead to the acquisition of
further mutations in CSCs, making them more complex and
potentially drug resistant. However, this may not be pragmatic in highly proliferative malignancies such as AML, and
the concurrent use of CSC- targeted therapies and “debulking” chemotherapy would therefore be more appropriate. It
has also been proposed to use CSC- targeted treatment in the
post- remission setting, either as part of the consolidation
regimen or as long- term maintenance strategy in order to
eradicate residual CSCs.
targeted therapies can be
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Additionally, caution should be exercised in applying the
same clinical endpoints used to measure the efficacy of
standard cancer therapies, as they could potentially result in
significant underestimates of ultimate clinical benefit, leading to erroneous conclusions about their efficacy. To make
an accurate assessment of response rates, long follow- up
periods are required; however, in the short term, clinical
endpoints such as progression- free and overall survival may
be more meaningful.
Validation of CSC- targeted therapies in the invivo setting
is also paramount, as stemness is a functional definition.
This would require having reliable methods to isolate CSCs,
which poses an additional challenge, as CSCs can be phenotypically unstable. Finally, a rational selection of combination therapies is required in order to maximize efficacy and
reduce the emergence of resistance.
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He, J., Nguyen, A.T., and Zhang, Y. (2011). KDM2b/JHDM1b, an
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Other CSC- targeted therapies
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chondrial oxidative phosphorylation eradicates therapychronic myeloid leukemia stem cells. Nat. Med. 23: 1234–1240.
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2 depend-
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Chapter25
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CAR- T cell therapy
Jude Franklin1, Prateek Pophali2, Zachary Jackson1, David Wald1,
andDavidAvigan
1
Department of Pathology, Case Western Reserve University, Cleveland, OH, USA
2
Division of Hematology and Hematological Malignancies, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
2
History of CAR- T cell therapy, 369
CAR structure and function, 370
CAR- T cell manufacturing, 372
CAR- T disease- specific clinical data, 374
Over the past decade, discoveries in the field of cancer
immunobiology and the elucidation of mechanisms of
tumor- directed tolerance have provided the critical background necessary for the development of clinically effective
immunotherapy. Chimeric antigen receptor T (CAR- T) cells
have poignantly demonstrated the potency of engineered
immune effector cells to selectively target and eradicate
malignant cells. CAR- T cell therapy utilizes an artificial
receptor that combines the antigen specificity of an antibody
with the intracellular signaling domains of T- cell receptors
(TCRs), such as CD3ζ, and co- stimulatory molecules (e.g.
CD28 & 4- 1BB). This design allows for the targeting of
tumor cells expressing a tumor- associated antigen (TAA) on
the cell surface by engineered T cells. Given its success in
hematological malignancies, CAR- T cell therapy is currently
being evaluated in a host of diverse contexts including solid
tumors and autoimmune disorders. Here, we discuss the history of CAR- T cell therapy, the structure, and function of the
CAR components, current methods, and considerations in
manufacturing CAR- T cells, the various malignancies that
are treated by CAR-
T therapy, and strategies that are being
tested to overcome intrinsic and extrinsic mechanisms of
resistance to CAR- T cell therapies.
History of CAR- T cell therapy
First designed in 1993 by Zelig Eshhar, the first- generation
CAR- T cells included CD3ζ as an intracellular signaling
domain without additional co- stimulatory domains (Eshhar
etal.1993). This design demonstrated limited efficacy, with
relatively poor activation and persistence of the T cells
Sources of CAR- T resistance, 379
Toxicities of CAR- T therapy, 382
CAR- T cells in the tumor microenvironment, 384
References, 387
observed in and/or cytokine release syndrome due to
supplementary treatments such as with high dose IL-
2 (Jensen
etal.2010; Büning etal.2010; Kershaw etal.2006). This was
followed by the development of second- generation CAR- T
cells targeting CD19 in lymphoproliferative diseases that
included the insertion of the costimulatory ligands CD28 or
4-1BB, which have demonstrated dramatic therapeutic efficacy
leading to FDA approval for a subset of patients with pediatric
acute lymphocytic leukemia (ALL), large cell, follicular and
mantle cell non- Hodgkin’s lymphoma (Maher et al. 2002;
Brentjens etal.2003). Similarly, CAR- T cell therapy targeting
BCMA in the context of 41-BB- mediated co- stimulation was
approved for patients with relapsed multiple myeloma. As outlined below, subsequent efforts have explored the targeting of
alternative antigens, incorporation of novel costimulatory
molecules, limiting CAR- T immunogenicity, enhancement of
CAR- T expansion and persistence while minimizing CAR- T
exhaustion, and modulation of the tumor immune microenvironment and native effector cell population. While therapeutic efficacy has been demonstrated hematologic malignancies,
T cells targeting solid tumors via mesothelin, GD2,
CARprostate- specific membrane antigen (PSMA), and mucin- 1
(MUC- 1) have demonstrated disappointing results (Marofi
etal.2021; Patel etal.2022). A greater understanding of mechanisms of resistance, including antigen escape, lack of CAR
persistence, functional exhaustion, and immunoregulatory
impact of the surrounding microenvironment, has led to nextgeneration strategies that will extend the efficacy of CAR- T
therapy to new targets and contexts, which will be discussed in
future sections. In Table25.1, there is a summary of studied
CAR- T cells and their respective success and failures (Sterner
and Sterner2021; St Martin etal.2023).
Molecular Hematology, Fifth Edition. Edited by Drew Provan and Hillard M. Lazarus.
© 2024 John Wiley & Sons Ltd. Published 2024 by John Wiley & Sons Ltd.
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369
370 Molecular Hematology
Generations of CAR-T cells
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Table25.1 CAR- T cell therapy successes andareas
forimprovement
CAR- T cell
type
CD19 CAR-
BCMA CAR- T Multiple
CD22 CAR- T Promising
HER2 CAR- T Glioblastoma
Successes/
disease type
T B- cell
malignancies
myeloma,
favorable
safety profile
efficacy in
adults with
ALL and
lymphoma
treatment
Failures/areas of
improvement
Toxicities, solid tumor efficacy,
immune inhibition, and
resistance in B- cell
malignancies
Disease- resistant mechanism
of downregulation of
BCMA, antigen escape, so
tandem CARs to target
multiple scFvs are used
Downregulation of antigen,
relapse from CAR- T therapy
failure, immunosuppressive
environment, onoff- tumor toxicities, severe
cytokine production
Limited penetration, but dual
targeting of HER2 and
IL13a2leads to improved
anti- tumor efficacy
target but
CAR structure and function
Chimeric antigen receptors are composed of an antibodyderived single- chain variable fragment (scFv), hinge domain,
transmembrane domain, and one or more intracellular stimulatory domains. Second- and third- generation CAR- T cells
are differentiated by the number of co- stimulatory domains.
Fourth- generation CARs (T cells redirected for antigenunrestricted cytokine- initiated killing- TRUCKS), are
designed to secrete cytokines such as IL- 7, IL- 12, IL- 15, IL18, or IL- 23, as a result of CAR signaling (Chmielewski and
Abken2020). Here, we discuss the impact of the individual
CAR components on the function of the CAR (Figure25.1).
Extracellular scFv and linker
The extracellular scFv of the CAR is composed of antibodyderived variable heavy and variable light chains connected
by a flexible linker or spacer. This element of the CAR is what
allows MHC- independent recognition of surface tumorassociated antigens (TAA). The primary considerations that
go into the design of the scFv are the choice of targeted TAA
and the corresponding specificity/affinity of the scFv (Liu
et al. 2015; Caruso et al. 2015; Ghorashian et al. 2019;
CAR-T
cell
First
Antigenbinding
domain
Hinge and spacer
Transmembrane
domain
Intracellular
signalling
domain
Figure25.1 Structural comparison of CAR- T cell generations. The first- generation CAR- T cells include CD3z as an intracellular signaling domain
without additional co- stimulatory domains. The second generation has one costimulatory domain, either CD28 or 4-1BB. CD28 is shown to have
a more proliferative response (increased expansion, decreased persistence) with an effector memory population, whereas the 4-1BB domain
results in better persistence of the CAR- T cells. CD28has also been theorized to lead to increased exhaustion. For this reason, third- generation
CARs are made using both domains. Further generations improve upon the signal process with cytokine inducers (IL- 7, IL- 12, IL- 15, IL- 18, or IL- 23)
to improve proliferation and differentiation. Each generation improves the proliferation and the signaling response with these edits to the
intracellular domains. Source: Made using BioRender.
generation
VL
VH
scFv
CD3-ζ CD28
Second
generation
Co-stimulatory
domains
Third
generation
41BB
Fourth
generation
Cytokine
inducer
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CAR- T cell therapy 371
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Chmielewski et al. 2004). Higher affinity binding may
enhance efficacy but could result in hyperstimulation and
earlier progression to an exhausted phenotype. In addition,
the consequence of off- target effects may be accentuated if
binding to lower- density antigen targets is increased. As the
scFv sequence is often derived from a murine antibody
sequence, this results in increased immunogenicity and
risk of rejection by the host immune system and the potential for rapid clearance of the CAR- T cells (Jensen
etal.2010). One area of investigation is the use of humanized scFvs to reduce risk of rejection and improve CAR- T
persistence (Sommermeyer etal.2017).
Choice of CAR- T targets is impacted by the heterogeneity in the expression of the TAA, the associated biologic
relevance for oncogenesis, and the relative prominence of
antigen- negative variants. Ideally, antigen expression would
be unique to the malignant population, or, alternatively,
expression is confined to a target population such as B cells
(CD19) or plasma cells (BCMA) whose function may be
supported, such as IVIG replacement. Choice of CAR- T
antigens has also been explored in the setting of differential
levels of TAA expression on malignant as compared to normal tissue, as exemplified by mesothelin (Hassan and
Ho2008). Investigators have also examined the targeting of
multiple antigens concurrently such as CD19, CD20, and
CD22in lymphoma. In addition, the shedding of tumorassociated antigens into the serum may result in saturation
of CAR- T receptors and reduce cytotoxicity at the tumor
site. Gamma secretase inhibitors have been employed to
limit shedding of BCMA and enhance CAR- T- mediated
killing of myeloma targets. Toxicity may arise from activation of effector cells with nonspecific, off- target impact on
the surrounding cell populations or via systemic immunemediated events. Alternatively, targeting of normal tissues
exhibiting antigen expression may be observed as manifested by potential neurotoxicity due to CD19 expression in
the brain (Hirayama and Turtle2019). In summary, scFv,
CAR expression, and target antigen density can play a significant role in CAR-
T cell function by influencing the
amount of antigen- dependent and independent signaling
(Greenman et al. 2021; Gomes- Silva et al. 2017; Long
etal.2015). Thus, in the case of mesothelin, specificity may
only truly be obtained by appropriately optimizing the
expression and affinity of the scFV for the antigen density
on the target.
Unlike the choice of scFv and target antigen, CAR linkers
have been given little attention. Most CAR linkers are composed of serine and glycine repeats of 15–20 amino acids
and are intended to serve little function beyond connecting
the variable chains without disrupting their folding.
Surprisingly, a recent study observed disparate results in
two clinical trials despite near- identical CAR designs
besides linker length, the difference being one had 5 amino
acids while the other 20 (CAR linker length modulates
targeted CAR- T- cell efficacy in ALL 2021). In the
CD22follow- up work, it was determined that the length of the
linker was the defining difference, with the shorter linker
causing homodimer formation and clustering on the cell
surface leading to increased signal transduction and cytotoxic function. Therefore, it was noted how minor alterations to CAR design can have unanticipated effects.
Hinge domain
The hinge domain of CAR- T cells is thought to provide the
flexibility necessary for the scFv to overcome steric hindrance and bind to target antigen. The hinge domains typically used in CAR- T cells are derived from CD8α, CD28,
immunoglobulin G1 (IgG1), or IgG4, which vary significantly
in length, flexibility, and composition. The choice of hinge
domain heavily impacts epitope recognition, CAR signaling
threshold, and the strength of CAR activation, depending on
the chosen antigen and epitope (Zhang etal.2021; Fujiwara
et al. 2020; Guest et al. 2005; Hudecek et al. 2015; Qin
et al. 2017). For example, membrane- proximal epitopes
require hinges of greater length, and membrane- distal
epitopes requiring hinges of shorter length (Fujiwara
etal.2020; Guest etal.2005; Hudecek etal.2015). In some
cases, the inclusion of a hinge domain may even be unnecessary (Qin etal.2017).
Transmembrane domain
The transmembrane domain of the CAR facilitates its
anchoring in the cell membrane. As such, the choice of a
transmembrane domain has a direct impact on CAR expression levels, CAR stability, and possibly immune synapse formation. This, in turn, significantly impacts levels of CAR
antigen- dependent and independent signaling (Fujiwara
et al. 2020; Bridgeman et al. 2010). The transmembrane
domains used in CAR-
T cell therapy are not well studied so
options are limited to those derived either from CD28 or
CD8, as CD3 was found to have decreased stability
(Bridgeman etal.2010). A study comparing the CD28 and
CD8 transmembrane domains paired with their respective
hinge domains observed little effect on CAR expression levels but increased TNFα and IFNγ cytokine production as
well as susceptibility to activation- induced cell death with
the CD28 hinge and transmembrane domain compared to
those of CD8 (Fujiwara etal. 2020; Alabanza et al.2017).
While not tested clinically, one study suggested improvement of a third- generation CAR via utilization of the transmembrane domain linked to the membrane proximal
intracellular domain (Guedan etal.2018). Still, future work
may reveal superior designs regarding selection of transmembrane domains.
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Intracellular co- stimulatory domains
The intracellular co- stimulatory domains of CARs have
received significant attention for their undeniable contribution to CAR function by driving signal transduction upon
ligand binding. First- generation CARs only contained CD3ζ
or (rarely) Fc receptor γ signaling domains and were overall
ineffective in generating T- cell responses with reduced
expansion, persistence, IL- 2 production, and clinical efficacy
(Eshhar et al.1993; Jensen et al. 2010; Büning et al.2010;
Brocker and Karjalainen 1995). Second- generation CARs
incorporated CD28 or 4- 1BB signaling domains in addition
to CD3ζ and were equally successful in improving the antitumor response (Maher etal.2002; Imai etal.2004). While no
direct clinical comparison of CD28 and 4- 1BB exists, preclinical and clinical evidence suggests that CD28 facilitates
more rapid T cell kinetics, a propensity for T cell exhaustion,
decreased persistence, and greater glycolytic metabolism in
comparison to 4- 1BB in CAR- T cells (Long et al. 2015;
Kawalekar et al. 2016; Zhao et al.2015). CD28 and 4- 1BB
signal through phosphoinositide 3- kinase (PI3K) and tumor
necrosis factor receptor–associated factors (TRAFs), respectively. Though, characteristics also thought to drive their differences are the increased level of recruitment of
lymphocyte- specific protein tyrosine kinase (Lck), the
increased degree of antigen- independent phosphorylation,
and the decreased level of phosphatase recruitment in CD28
CAR- T cells (Sun et al.2020). Interestingly, a recent study
shows that CD28 CAR- T drives the cells to become
exhausted, while the 4-1BB costimulatory domains result in
more dysfunctional phenotypes, showing that there are
divergent signaling patterns as a result of different costimulatory domains (Selli etal.2023). Alternative co- stimulatory
domains with positive results that are yet to be tested clinically include CD27, OX40, inducible T cell costimulator
(ICOS), myeloid differentiation primary response 88
(MYD88), and CD40 (Hombach etal.2012; Mata etal.2017;
Song and Powell2012; Guedan etal.2014). Third- generation
T cells incorporate three co- stimulatory domains in
CARcomparison to two with the theory of further simulating T
cell activation (Pulè et al. 2005). Combinations that have
been tested include CD3ζ/CD28/4- 1BB or CD3ζ/CD28/
OX40 and the results vary in their efficacy. In a model of
lymphoma, CD3ζ/CD28/4- 1BB CAR- T cells showed superior anti- tumor efficacy to the second- generation CARs
(Zhong et al. 2010). Whereas, in models of leukemia and
pancreatic cancer, third- generation CARs performed inferior to second- generation (Abate- Daga et al.2014; Milone
et al. 2009). Moreover, early- phase clinical trials utilizing
third- generation CAR- T cells have shown comparable efficacy to second- generation CAR- T cells (Enblad etal.2018).
In another trial, CD3ζ/CD28/4- 1BB CAR- T cells expanded
28 times more than CD3ζ/CD28 second- generation CAR- T
cells, although clinical efficacy was not compared (Gomes da
Silva etal.2016).
CAR- T cell manufacturing
There are many considerations related to the methods used
for CAR- T manufacturing. Subtle changes in manufacturing
methods can result in significant changes in clinical efficacy
due to alterations in the underlying T cell composition (i.e.
percentage of CD4 and CD8 T cells), the percentage and
level of CAR expression, CAR- T cell differentiation, product
purity, and safety/toxicities associated with the therapy. The
methods that are most likely to influence clinical efficacy are
cell isolation, construct integration, expansion duration, and
cytokine stimulation. Furthermore, significant consideration
must be given to the facilities, cost, and time needed to manufacture CAR- T cells. While most drugs are readily available
and manufactured in bulk, CAR- T cells are typically autologous (i.e. patient- derived) cells that require time and specialized good manufacturing practice (GMP) facilities/personnel
to manufacture. Autologous CAR- T generation is a multi-
kapheresis, transgenic expression of CAR accomplished via
viral transduction, cellular expansion ex- vivo, confirmation
of sterility, and lymphodepleting chemotherapy conditioning before infusion of the CAR- T cells. This process usually
takes weeks and patients may require bridging therapy for
disease control until CAR- T cell administration. The use of
autologous CAR- T cells is dependent on the functional competency of the T cell substrate that may be impacted by disease status and prior cytotoxic therapies. Allogeneic cell
CAR- T cells have been explored as providing an “off- theshelf” source of cell therapy from normal donors without the
complex logistics of autologous cell production. However,
persistence of allogeneic product may be limited by immune
rejection and interactions between the CAR- T and host
immunity are blunted. At present, the methods used to manufacture CARthe global scalability and accessibility of CAR- T cells, and
innovations in the technology for these methods are being
explored (Figure25.2).
Cell isolation method
Peripheral blood mononuclear cells are isolated by leukapheresis and leukocytes may be enriched for T cells, prior to activation, often via bead- bound antibody selection of CD3 or CD4
and CD8 expression. In other cases, the CD4:CD8 T cell ratio
is altered to improve consistency, but while the inclusion of
CD4 T cells is thought to improve efficacy, the impact of maintaining a CD4:CD8 ratio remains unclear (Sommermeyer
etal.2016; Turtle etal.2016). Similar strategies to specifically
T cells are considered significant barriers to
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