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by the cancer treatment. Autologous mesenchymal stem cells generally do not appear to pose
a risk for immune rejection (Chen et al., 2023). However, there is conflicting information in
the literature with autologous iPSCs. Zhao et al. (2011) demonstrated that iPSC-derived ter-
atomas, but not ESC-derived teratomas, implanted into syngeneic mice were immune
rejected, and displayed T-cell infiltration and tissue necrosis. They further hypothesized
that this difference was due to incomplete reprogramming of the iPSCs. In support of this,
other researchers did not elicit an immunogenic response when syngeneic mice were
implanted with either (a) in vivo-differentiated tissues from iPSC chimeric mice (Araki
et al., 2013), or (b) iPSCs that were differentiated into embryoid bodies or representative
cell types from each of the embryonic layers (Guha et al., 2013). In addition, nonhuman pri-
mates implanted with autologous iPSC-derived dopamine neurons showed lasting engraft-
ment for 2 years, without immunosuppression (Emborg et al., 2013). Furthermore, a
patient with age-related macular degeneration administered autologous iPSC-derived retinal
epithelial cells showed long-term survival of transplanted cells for 25 months without im-
mune suppression (Mandai et al., 2017). However, in humanized mouse models, autologous
human iPSC-derived smooth muscle cells, but not iPSC-derived retinal pigmented epithelial
cells were immunogenic (Zhao et al., 2015). Collectively, the data indicate that undifferenti-
ated iPSCs may be more immunogenic than differentiated iPSCs, but this could differ be-
tween cell types. Regardless, the use of autologous stem cells are also limited by the length
of time it can take to generate the cells, the presence of genetic defects within a patient’s
own cells, and/or cost. In fact, it has been estimated that the generation of an autologous
iPSC cellular product for one patient can take several months, and cost almost $800,000
(Doss and Sachinidis, 2019).
To address the limitations of autologous stem cells, allogeneic stem cells are considered.
Allogeneic stem cells are collected from a single source but transplanted into many different
patients. These “off-th e-shelf” therapies have greater potential for immune rejection. Immune
rejection of allogeneic stem cells can occur because of the innate or adaptive arms of the im-
mune system. Acute graft rejection, which happens within days to weeks after implantation,
occurs because of direct cytotoxicity that has been initiated by CD8þ cytotoxic T lymphocytes
after interacting with the major histocompatibility complex (MHC) class I molecules on the
stem cells. By comparison, chronic graft rejection generally occurs because MHC class II mol-
ecules on the stem cells are recognized as foreign by CD4þ effector T cells, which in turn ac-
tivates and enhances the cytotoxic and humoral responses (Chen et al., 2023). The potential
for immune rejection can by influenced by several factors, which includes the following:
(1) An increased presence of undifferentiated iPSCs may increase the risk for immune rejec-
tion. (2) The specific cell type derived from the iPSC. For example, iPSC-derived smooth mus-
cle cells can lead to an intense immune response, but iPSC-derived retinal epithelial cells,
cartilage and neural crest cells have low potential for immunogenicity. This is mostly because
they have less expression of MHC molecules. (3) The site where the stem cells are trans-
planted. For example, iPSCs implanted by SC or IM injection elicited an immune response
in mice but implantation into the kidney capsule did not. (4) The accumulation of mutations
in mitochondrial DNA during the re-programming or long-term culture phase can cause the
formation of neoantigens that lead to an immune reaction and ultimately, rejection of the
stem cells (Qiao et al., 2020).
10. Considerations for nonclinical studies conducted to support the development of cellular therapies326
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Immune rejection can be ameliorated by suppression of host immunity; human leukocyte
antigen (HLA) matching; and/or generating hypoimmune cells. Global suppression of the
host immune system with coadministered drugs (such as calcineurin inhibitors, mTOR inhib-
itors, de novo purine synthesis inhibitors, and glucocorticoids), is a common way to evade
rejection of allogeneic stem cells. However, prolonged immunosuppression increases the
risk for tumors, kidney failure and severe infections. Furthermore, these drugs can suppress
immune effector function, which make them incompatible for CAR-T and CAR-NK cell ther-
apies. Another way to suppress the host immune system is to deplete lymphocytes that
mediate rejection, prior to infusion of the cell therapy. This preconditioning can be accom-
plished with drugs such as cyclophosphamide and fludarabine, busulfan, or with antibodies
against antigens on host lymphocytes such as CD52. However, careful timing is essential
when using this method because the preconditioning agent must be administered and cleared
before the cell therapy treatment can occur. In addition, precondition may also be associated
with toxic side effects that could limit the number of preconditioning doses. For example,
busulfan, which is commonly used as a preconditioning agent for transplanting hematopoi-
etic stem cells is known to cause infertility, pulmonary fibrosis, and changes in skin pigmen-
tation (Guo et al., 2019).
A second strategy to evade immune rejection is to match human leukocyte antigen (HLA)
haplotypes of the donor and the recipient. In humans, the MHC system is also known as the
HLA system. There are three HLA regions; class I, class II, and class III. The main function of
HLA class I gene products (HLA-A, -B, and -C) is to present endogenous peptides to respond-
ing CD8þ T cells. Class II coded molecules HLA-DR, -DP, and -DQ, process exogenous pep-
tides for presentation to CD4þ helper T cells. The class III region contains genes that encode
for immune regulatory molecules, e.g., tumor necrosis factor (TNF), complement factors C3,
C4, and C5, and heat shock proteins (Cruz-Tapias et al., 2013). Since T cell self-recognition of
HLA on allogeneic cells is a critical step for preventing the initiation of cytotoxicity and anti-
body formation, the generation of iPSCs from donors that HLA-match that of the recipient,
should reduce the potential for immune rejection. MHC matching to reduce allogeneic immu-
nogenicity or immune rejection and/or enhance engraftment, has been observed nonclini-
cally in nonhuman primates administered iPSC-derived cardiomyocytes (Kawamura et al.,
2016; Shiba et al., 2016), or dopaminergic neurons (Morizane et al., 2017). HLA matching
to reduce the risk of immune rejection has also been demonstrated clinically. Sibling bone
marrow transplants where HLA class I and II genes of the donor were matched to those of
the patient, had a reduction in graft rejection. However, iPSC transplant recipients that
were HLA-matched to the donors can still have immune rejection because of minor antigens
that are presented on the cell surface HLA. Furthermore, the risk for rejection increases with
the num ber of mismatched HLA alleles.
In support of the HLA-matching strategy, banks of allogeneic iPSCs with genetically
defined MHC/HLA have been proposed. However, HLA matching is challenging to execute
because of the high degree of HLA polymorphisms in the general population. It has been esti-
mated that iPSCs generated from w140 to 150 unique donors would be sufficient to HLA
match w90%e93% patients from Japan (Okita et al., 2011) or the United Kingdom (Taylor
et al., 2012). Unfortunately, more donors are predicted to be needed for more diverse popu-
lations, like those in the United States (Wang et al., 2022).
Development challenges 327
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A third strategy to evade immune rejection is to use gene editing techniques to create
hypoimmune cells. Using Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)/CRISPR associated protein 9 (Cas9)) gene-editing system, the beta 2-
microglobulin (B2M) gene, and Class II Major Histocompatibility Complex Transactivator
(CIITA) gene were knocked out of mouse or human iPSCs, to cause them to lose their immu-
nogenicity (Mattapally et al., 2018; Gornalusse et al., 2017). These hypoimmunogenic iPSCs
retained their pluripotent stem cell potential and differentiation capacity. B2M is a structural
component of MHC class I (and HLA class 1 for human cells), and CIITA is the master regu-
lator of all class II molecules. Knockout of B2M and CIITA eliminates the cell surface expres-
sion of all class I and II molecules and may render the edited cells unrecognizable by host
CD8þ and CD4þ T cells. However, the deletion of HLA expression leaves the cells vulnerable
to NK cell killing. To address this issue, cells can be edited to: (1) also express HLA-E to
inhibit NK cell-dependent lysis through its binding to CD94/NGK2A (Gornalusse et al.,
2017); or (2) overexpress integrin-associated protein, CD47, to enhance the “don’t eat me”
signal (Deuse et al., 2019). CD47 is ubiquitous membrane protein that can interact with
several cell surface receptors to inhibit phagocytosis (Jaiswal et al., 2009). Endothelial cells,
smooth muscle cells, and cardiomyocytes derived from mouse and human iPSCs with inac-
tivated MHC class I and II and overexpressed CD47, consistently evaded immune rejection in
fully MHC-mismatched allogeneic recipients, and survived long-term, without the use of
immunosuppression (Deuse et al., 2019). Thus, the use of hypoimmune iPSCs represents a
promising strategy to evade immune rejection.
Challenges associated with CAR-T cell therapies
Although treatment with CAR-T cells has produced remarkable clinical responses with
certain subsets of B cell leukemia or lymphoma, many challenges limit the therapeutic effi-
cacy of CAR-T cells in solid tumors and hematological malignancies. Barriers to effective
CAR-T cell therapy include severe life-threatening toxicities, modest antitumor activity, anti-
gen escape, restricted trafficking, and limited tumor infiltration. This section will focus on the
toxicities.
Toxicities mainly arise from crosstalk between activated CAR-T cells; and then macro-
phages provoke an inflammatory reaction that leads to cytokine release syndrome (CRS)
(Fig. 10.2). This response can also activate endothelial cells, which leads to neurotoxicity
known as immune effect or cell-associated neurotoxicity syndrome (ICANS). Toxicities arising
from healthy tissue damage can not only result from off-tumor on-target or of f-target of CAR
recognizing its target on normal cells but also through recognition of an unrelated peptide by
engineered T-cell receptor (TCR) or CAR-T (cross-reactivity). Other toxicology issues that are
connected to CAR T therapy include: (1) hematological toxicities, arising from the adminis-
tration of lymphodepleting regimens; (2) allogeneic CAR T cells that have the potential to
induce graft-versus-host disease when T cells interact with foreign MHC molecules on host
cells, (3) viral integration, and risk for mutagenesis; and (4) T-cell clonal dominance.
In the past decade, several efforts have been dedicated to the establishment of more pre-
dictive and reliable models to understand CAR T toxicity. In this chapter we highlight the
advantages, as well as the limitations, of current models and propose measures (Table 10.2)
to have nonclinical models fit for purpose with respect to engineered T-cell toxicity profiling.
10. Considerations for nonclinical studies conducted to support the development of cellular therapies328
III. Specialty biologics and indications
CRS and neurotoxicity
CRS is defined by the American Society for Transplantation and Cellular Therapy (ASTCT)
consensus group as the “supraphysiologic response following immune therapy that results in
activation or engagement of endogenous or infused T cells” (Lee et al., 2019). The cytokine
storm that results from CRS can lead to a variety of symptoms, ranging from mild to severe.
The symptoms typically appear within the first few days after cell infusion and can include
fever, chills, headache, nausea, vomiting, and muscle pain. In severe cases, patients may
experience hypotension (low blood pressure), tachycardia (rapid heart rate), and respiratory
distress (Fig. 10.2). The release of cytokines can also cause multiorgan dysfunction, such as
hepatic and renal failure, and in severe cases, it can be fatal, without timely intervention.
In the most severe form, CRS shares many features with hemophagocytic lymphohistiocyto-
sis and macrophage activation syndrome (June et al., 2018).
CRS occurs when the immune cells that are infused into the patient become activated and
release large amounts of cytokines, which are signaling molecules that regulate the immune
system (Suntharalingam et al., 2006). Immune cells, such as T cells, can become activated
when they recognize and bind to speci fic targets, such as cancer cells or infected cells.
FIGURE 10.2 Overview of the main adverse events associated with engineered T cells. CAR, chimeric antigen
receptor. Created with Biorender; Modified from Donnadieu et al. (2022) and Yang et al. (2023).
Development challenges 329
III. Specialty biologics and indications
Once activated, the T cells can start to proliferate and release cytokines, which can then acti-
vate other immune cells, creating a positive feedback loop that amplifies the cytokine
response ( June et al., 2018).
The severity of the CAR T cell-associated CRS correlates wi th tumor burden (Brentjens
et al., 2013; Maude et al., 2014; Neelapu et al., 2018), which can be more severe than the
flu-like symptoms commonly observed with TIL- and TCR-based therapies (Kalos et al.,
2011). Although CRS was an expected toxicity with T cell immunotherapy, unexpected
neurologic complications: depressed level of consciousness, seizure, paresis, elevated intra-
cranial pressure, cerebral edema, ranging in severity from mild to life-threatening, have
also been reported across different clinical studies with CD19and BCMA-specific CAR
T cells. The neurologic toxicities described with CD19-specific CAR T cells have been largely
reversible. It is not known whether the cerebral edema resulting from CAR-T therapy is an
extreme manifestation of CRS or whether there is a separate mechanism of action. In support
of the latter, there is evidence for endothelial injury, perhaps related to inflammatory cyto-
kines, contributing to the onset of neurotoxicity. The mechanisms (Fig. 10.3) underlying
T cell immunotherapy-mediated CRS and cerebral edema are poorly understood, in part
because the field lacks informative animal models to study these important toxicities.
FIGURE 10.3 CAR T cell therapy is associated with cytokine release syndrome and neurotoxicity. Cytokine
release syndrome has occurred with CAR T cells targeting CD19 or BCMA. When the CAR T cell engages surrogate
antigens, it releases a variety of cytokines and chemokines. Macrophages and other cells of the innate immune system
also become activated and contribute to the release of soluble mediators. CAR T cells are routinely observed in ce-
rebrospinal fluid, and the cytokines may increase permeability to soluble mediators and permit increased trafficking
of CAR T cells and other lymphocytes to central nervous system parenchyma. ALT, alanine aminotransferase; AST,
aspartate aminotransferase; IFN, interferon. Adapted from June et al. (2018).
10. Considerations for nonclinical studies conducted to support the development of cellular therapies330
III. Specialty biologics and indications
Models for understanding CRS and neurotoxicity
Utility of biomarkers for predicting CRS and neurotoxicity
The ability to predict which pa-
tients will become critically ill with severe CRS is vital to the development of CAR T-cell
therapy (Teachey et al., 2106). Biomarkers can objectively measure and evaluate biological
or pathogenic processes and/or indicators of pharmacological resp onses to a therapeutic
intervention (Biomarkers Definitions Working Group, 2001) and are an essential compo-
nent of preclinical safety assessment of CAR-T cells. The identification of predictive
biomarkers may be crucial for the s election of patients at risk of developing severe toxicities
who might benefit from early therapeutic intervention. The immune monitoring for CRS in
patients treated w ith CD19 CAR T cells includes serum biomarkers su ch as MCP- 1, SGP130,
interferon gamma, IL-1, eotaxin, IL-13,IL-10, macrophage inflammatory protein-1 alpha
(Hay et al., 2017; Teachey et al., 2016); as well as IL-6, IL-15, and TGF-b28 29 as independent
predictors in statistical models ass essing risk of CRS and neurotoxicity, respectively (Ro ssi
et al., 2018; Turtle et al., 2016a,b). However, an accurate and reliable method to predict
which patients will become critically ill with severe CRS could not be located in the
literature.
Animal models for CRS and neurotoxicity assessments Mice, nonhuman primates, and dogs
are all potential animal models to evaluate CRS and neurotoxicity. These models have the
advantage of recapitulating the complex crosstalk between CAR T cells and host immune
cells; and in dogs, offer the possibility to test engineered T-cell efficacy with natu rally occur-
ring tumors. In addition, these models all have a functional myeloid compartment, which
allotransplantation studies in various animal models have demonstrated is necessary to
trigger CRS (Donnadieu et al., 2022). Syngeneic mouse strains, that are comprised of intact
immune cells and murine CAR T cells have been employed to predict CRS and ICANS
(Fig. 10.4). Although CRS was not observed after infusion of human CAR T cells into immu-
nodeficient NSG mice, it was reported in SCID-beige mice, which feature a less compromised
myeloid compartment. By using the SCID-beige model, it was possible to prove that this
FIGURE 10.4 Overview of the models and tools used to predict engineered T cell-associated toxicity. Adapted
from Donnadieu et al. (2022).
Development challenges 331
III. Specialty biologics and indications
reaction is triggered by resident macrophages due to both contact-dependent and cytokine-
related mechanisms, such as nitric oxide together with IL-1 and IL-6 release (Donnadieu
et al., 2022). In contrast to other models, humanized SGM3 mice were also able to recapitulate
neurotoxic manifestations, which in this case cannot be ascribed to on-target, off-tumor reac-
tions against mural cells but rather, might be connected to CRS-related inflammatory reac-
tions (Norelli et al., 2018). Alternatively, nonhuman primates are excellent large animal
models to investigate CAR-T cell toxicity because they have an immune system similar to
humans, and they closely recapitulate CRS and ICANS development. However, the draw-
back is that primate models often require autologous CAR T cells and often do not have natu-
rally occurring tumors. Finally, because of biological similarities between canine and human
cancer, dogs with naturally occurring tumors offer a model to test engineered T-cell effects
(Taraseviciute et al., 2018; Künkele et al., 2017; Berger et al., 2015; Mochel et al., 2019).
In vitro models for biomarker assessment In vitro coculture models, that consist of
monolayers of cells expressing the target antigen, assess the antitumor efficacy of “engi-
neered” immune cells in clinical trials such as CAR-T cells, CAR-natural killer (NK) cells,
and CAR-macrophages, by using sensitive and reproducible in vitro cytotoxicity assays.
They are the chromium (51Cr)erelease assay (51Cr assay), the luciferase-mediated biolumi-
nescence imagi ng (BLI) assay, the impedance-based assay, and the flow cytometry assay
(flow assay). However, these assays alone, are not considered appropriate to predict CRS
(Kiesgen et al., 2021)(Fig. 10.3). To address this shortcoming, macrophages, which play an
important role in the pathogenesis of CAR-T-mediated CRS, are included in the cocultures
of target and CAR-T cells (Liu et al., 2020, 2021; Staedtke et al., 2018; Norelli et al., 2018).
This inclusion improved the ability of in vitro cytotoxicity assays to predict CRS by
measuring novel soluble biomarkers contained within supernatants from these in vitro sys-
tems. Furthermore, it facilitated investigations into mechanistic causes of CRS; which led
to the finding that pore-forming protein gasdermin E (GSDME) during tumor cell pyroptosis,
contributes to CRS (Liu et al., 2020). CAR T cells release granzyme B, which activates caspase
3 and cleaves GSDME in target tumor cells and causes pyroptosis. Pyroptosis by GSDME
leads to activation of caspase 1 and GSDMD in macrophages, which is critical for triggering
CRS. Interestingly, elevated levels of GSDME in cance r patients positively correlated to CRS
severity. CRS induced by T-cell-activating therapeutic agents, including CAR-Ts, was also
accompanied by a catecholamine surge in vitro and in mice (Staedtke et al., 2018). Pharma-
cologic catecholamine blockade with metyrosine protected mice from lethal complications of
CRS. Collectively, mechanistic insights obtained via in vitro assessment of CRS may help
identifying biomarkers directly correlating to CAR-T cytotoxicity, as well as aid in pharma-
cologic reversal of CRS without impairing therapeutic response.
On-target and off-target, off-tumor toxicity
Since, target antigens are often expressed on both tumor cells and healthy tissues, there is
concern for on-target, off-tumor toxicity associated with CAR-T cell therapy. Accessibility,
breadth of expression of the target antigen across various target organs, and nature of the target
tissue can influence the severity of toxic manifestations (Ragoonanan et al., 2021). Examples of
toxicities, of various severities include: (1)controllable B-cell aplasia associated with the admin-
istration of CD19 CAR-T cells (Ragoonanan et al., 2021); (2) secondary
10. Considerations for nonclinical studies conducted to support the development of cellular therapies332
III. Specialty biologics and indications
hypogammaglobulinemia observed after administration of BCMA CAR-T cells (Zhou et al.,
2020); (3) liver toxicity, characterized by dose-limiting liver enzyme elevations observed
following administration of CAR T cells targeting carboxyanhydrase-IX (Lamers et al., 2013);
(4) severe and fatal pulmonary toxicity, which may involve the recognition of low levels of
ERBB2 on lung epithelial cells, observed with the administration of HER2 CAR T cells (Morgan
et al., 2010).
Long-term B-cell depletion is the most described on-target, off-tumor toxicity (Fig. 10.2)in
response to CD19 CAR T-cell trials. B-cell depletion also contributes to hypogammaglobulin-
emia; increases the risk of infection; and may impact the response to vaccinations (Walti et al.,
2021). For example, decreased seroprotection for vaccine-preventable infections was observed
in BCMA-CAR-T cell recipients, who had fewer pathogen-specific antibodies. In pediatric pa-
tients, B-cell depletion is generally managed with intravenous immunoglobulin supplemen-
tation. However, in adult patients, immunoglobulin supplementation is used only in patients
with recurrent bacterial infections.
Common (30%e40% of patients) postinfusion off-target off-tumor side effects include
cytopenias, such as CD4þ T-cell lymphopenia and neutropenia. Although the pathogenesis
is currently unclear, these findings can persist for more than 1 year, or more than 30 days,
respectively (Logue et al., 2021). The occurrence of these cytopenias is increased by (1) low
baseline ce ll counts; (2) prior allog eneic hematopoietic stem cell transplantati on (HSCT); (3)
impaired hematopoietic reserve; (4) bone marrow infiltration and chronic inflammation, as
indicated by higher baseline ferritin and C reactive protein levels and (5) alterations in
levels of the chemokine SDF-1 in the marrow microenvironment, that correlate with early
and late cytopenia observed after infusion of CD19 CAR-T cells in patients (Jain et al., 2020;
Fried et al., 2019). Neutropenia, thrombocytopenia, and anemia occur frequently (94%,
80%, and 51%, respectively) after CAR-T cell infusion, and with a biphasic nature. In
fact, hematologic toxicity occurs after 21 daysfromcellinfusionin93%ofpatients(Fried
et al., 2019). Late hematologic toxicity was more common in patients with high grade
CRS and in patients administered HSCT. Interestingly, since these events occur late after
the lymphodepleting chemotherapy and after resolution of CRS, alterations in SDF-1 levels
correlated with t he biphasic nature of late neutropenia (Fried et al., 2019). Off-target off-
tumor toxicity can also occur due to cross-reactive binding to a mimotope, which is a
similar but distinct epitope expressed on normal tissues. This cross-reactivity or ‘off-
target’ binding to cell surface proteins, is difficult to predict in nonclinical animal studies,
and can lead to serious adverse effects and in some rare instances death of a patient
(Donnadieu et al., 2022).
Models for on-target, off-target, and off-tumor toxicity
Models for on-target, off-tumor toxicity
Traditional preclinical models, including two-
dimensional cell culturing techniques and xenograft models in NSG mice, have failed to pre-
dict engineered T cell-associated adve rse events observed in the clinic. Recently, organoid
and animal models that include humanized, transgenic and syngeneic mouse models, as
well as primate or canine models, have been proven useful to predict several complications
observed in patients after chimeric antigen receptor T-cell infusion (Donnadieu et al., 2022;
Berger et al., 2015; Mochel et al., 2019; Schnalzger et al., 2019). This concern is heightened
for solid tumors when tumor-associated antigens are also expressed, even at low levels, on
Development challenges 333
III. Specialty biologics and indications
normal tissue, so-called “on-target” toxicity (Berger et al., 2015; Bonifant et al., 2016). In this
regard, syngeneic models (Fig. 10.4), are successful in investigating strategies t o overcome B -
cell aplasia induced by CD19 CAR T cells (Paszkiewicz et al., 2016). Administration of murine
CD19 CAR T cells to mice of different strains, including NSG, can cause blood brain barrier
leakiness and pericyte depletion, supporting the hypothesis that ICANS development in pa-
tients could also be the result of on-target, off-tumor recognition of CD19 on brain mural cells
(Parker et al., 2020). However, species-specific differences, especially for the assessment of
cognitive disorder, memory impairment, men tal status changes, paranoia, somnolence,
and stupor, is difficult to evaluate in mouse models. On the other hand, when t he target an-
tigen is expressed simi larly in both human and rodents and the antibody recognizes both
versions, human CAR T cells can be used in immunodeficient mice to study on-target,
off-tumor toxicity. Alternatively, on-target, off-tumor reactions can be studied in immuno-
competent transgenic mice that express a human tumor-associated antigen (TAA) alongside
its regulatory elements to mimic the spatiotemporal expressi on patterns seen in patie nts.
Thesemicecanbebredwithtumor-pronemiceordirectlygraftedwithTAAþ tumors to
test new CAR T-cell therapies.
For solid cancers, there is an unmet need for preclinical models to identify and select
suitable target antigens for CAR therapy. Schnalzger et al. (2019) describes a platform for
assessment of CAR-NK-92-mediated activity against patient-derived organoids in a 3D envi-
ronment, permitting more physiological analysis of effector cell recruitment and cytotoxicity
on a single organoid level. Using a panel of standardized CAR-NK-92 cells, this facilitated
rapid and individualized testing of efficacy for various test articles. In addition, potential un-
wanted toxicity to normal epithelia was also assessed.
Mitigation of on-target off-tumor toxicity observed in the liver with a CAIX CAR-T was
achieved by Berger et al. (2015) by administering a CAIX monoclonal antibody, which satu-
rated the liver at a single low dose of 5 mg, but left the CAIX antigen in renal cell carcinoma
metastasis accessible to the therapy at higher CAR-T cell doses.
Models for off-target off-tumor toxicity Minimizing the risk for initiating an inappropriate
immune response due to unanticipated off-target binding of CAR T cells to cell surface
proteins expressed on normal tissues is crucial for patient safety. Cell microarray technol-
ogies ( Fig. 10.4) have been developed to understand the off-target profile of CAR T cells
and demonstrate on-target specificity (Freeth and Soden, 2020). The Retrogenix Cell
Microarray Technology screens scFvs or whole CAR T cells for bindi ng against >4000
full-length proteins that are individually overexpressed in their native context in human
cells, to identify their interactions with both cell surface receptors and secreted proteins.
This platform, which was established in human cells and has a broad protein coverage,
can detect even low-affinity interactions with a high degree of sensitivity and specificity;
and can provide insights into potential off-target toxicities or soluble sinks for the thera-
peutic. If off-targets are identified, target cells expressing the off-target protein can be
used to assess potential off-target cytotoxicity and CAR T-cell activati on. This platform
is increasingly being u sed for CAR T-cell development (Adams et al., 2017), and data
from these studies have been included in regulatory submissions, including the biologics
license application for Novartis’sKymriah(Biologics license application (BLA) for
tisagenlecleucel, 2019).
10. Considerations for nonclinical studies conducted to support the development of cellular therapies334
III. Specialty biologics and indications
Graft-versus-host disease (GVHD) and rejection associated with allogeneic
engineered T cells
The utilization of allogeneic CAR T-cell products, which are generated using cells from
healthy donors, has the potential to overcome many of the limitations associated with autol-
ogous products. However, they also present their own set of challenges, including the possi-
bility of inducing GVHD (which is characterized by abdominal pain or cramps, nausea,
vomiting, and diarrhea) and the risk of immune-mediated reje ction by the host. Following
GVHD, there is often a relapse of malignancy. In fact, relapse of the original malignancy after
SCT now remains the most frequent cause of treatment failure and mortality. Approximately
40%e45% of recipients of HLA-identical siblings and approximately 35% of recipients of un-
related donor transplants will relapse with their original malignancy (Barrett and Battiwalla,
2010). The likelihood of GVHD developing is increased with increasing donor-recipient HLA
disparity, but this risk can be minimized through various approaches such as donor selection,
cell-type selection, T-cell depletion/selection, and gene editing. In particular, gene editing of
the endogenous ab T cell receptor (TCR), which typically involves the disruption of the T-cell
receptor alpha constant (TRAC) or T-cell receptor beta locus, reduces the risk of GVHD asso-
ciated with TCR recognition of allogeneic host tissue (Torikai et al., 2012). Additionally, the
use of gene-edited T cells deficient in the expression of CD52 has been explored in combina-
tion with alemtuzumab to maintain a prolonged conditioning regimen without affecting CAR
T product persistence; though concerns have been raised regarding prolonged lymphodeple-
tion regimens and the fate of T cells in the absence of their own TCR. Despite these chal-
lenges, clinical experience to date with allogeneic CAR, both virus-specific and from
allogeneic transplant donors, has shown antitumor effects with minimal GVHD risk (Torikai
et al., 2012; Qasim, 2019; Poirot et al., 2015).
Models for GVHD and rejection
To understand the mechanism whereby allogeneic CD19 CAR T cells may mediate anti-
lymphoma activity without causing a significant increase in the incidence of GVHD, Ghosh
et al. (2017) studied donor-derived CD19 CAR T cells in allo-HSCT and lymphoma models in
mice. Fig. 10.4 illustrates how these allogeneic mouse models with MHC-disparate genomes
are useful in studying graft-versus-host disease (GVHD). The models have revealed that acti-
vating both the exogenous CAR and the endogenous alloreactive TCR results in a decreased
risk of developing GVHD due to the loss of function and possible deletion of the transferred
T cells. Furthermore, xenoreactions in immunodeficient mice can serve as surrogate markers
for assessing the potential of engineered human T cells to cause GVHD in patients. In these
mouse models, GVHD scores have been established and applied based on several parame-
ters, including progressive weight loss, excessive expansion of T cells, ruffled fur, hunchback,
and T-cell infiltration of GVHD target organs. However, standard xenograft models are insuf-
ficient for assessing rejection potential, which can be evaluated instead in allogeneic mouse
models. More advanced alternatives, such as the human hematopoietic stem
cell-humanized SGM3 models, enable the measurement of GVHD potential as reactivity
against human hematopoietic cells from allogeneic CD34þ donors. Rejection potential can
be evaluated in these models by determining the time required to generate endogenous hu-
man T cells capable of rejecting allogeneic engineered T-cell products. Alternatively, studying
Development challenges 335
III. Specialty biologics and indications
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