Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5461_Библиотеки_им_академика_М_И_Перельмана
.pdf
Cell therapies involving immune cells (cellular immunotherapy) are typically used for
treating cancers. In fact, immunotherapy is now established as a fourth pillar of cancer treat-
ment, along with surgery, radiation, and chemotherapy (Maus et al., 2020 ). Genetically modi-
fied T cells are a novel form of immunotherapy, characterized by highly efficient and specific
targeting of tumor cells when compared with checkpoint inhibitors (Maus et al., 2020). By
applying sophisticated ex vivo culture and cellular engineering approaches to adoptive
T cell transfer (ACT), durable clini cal responses of otherwise treatment-refractory cancers
have been achieved (June et al., 2018). Cellular immunotherapies include approaches such
as: (1) Tumor infiltrating lymphocytes (TIL) therapy-T cells that have already infiltrated a tu-
mor are harvested, activated and expanded, and then a large number of activated T cells are
re-infused back into the patient. (2) Engineered T cell Receptor therapy-harvested T cells are
given a new T-cell receptor (TCR) to target specific cancer antigens, activated, expanded and
infused into the patient (3) Chimeric Antigen Receptor (CAR)-T cell Therapy-harvested
T cells are genetically altered to express the CAR, and following infusion into the patient,
CAR-T cells bind to naturally occurring antigens that are expressed on cancer cells, resulting
in T cell activation, recruitment of other immune system cells, and ultimately death of the
cancer cells. (4) Natural Killer (NK) cell therapies e Similar to CAR-T cells, CAR-NK cells
are genetically modified to express CARs that recognize a specific antigen uniquely expressed
or overexpressed by target cells.
At present, six autologous T cell products engineered to express a chimeric antigen recep-
tor (CAR-T) have been approved by the US Food and Drug Administration and other regu-
latory agencies with B-cell malignancies. These include Abecma, Breyanzi, Carvykti,
Kymriah, Tecartus, and Yescarta. Abecma and Carvykti target B-cell maturation antigen
(BCMA) and are indicated in the treatment of adults with relapsed or refractory myeloma af-
ter four or more prior lines of therapy, including an immunomodulatory agent, a proteosome
inhibitor, and an anti-CD38 monoclonal antibody. Kymriah, Yescarta, and Tecartus target
cluster designation 19 (CD19) and are indicated for the treatment of adults with distinct types
of B-cell malignancies, and in young adult patients with relapsed or refractory acute lympho-
blastic leukemia (ALL). Despite these successes, there are still challenges associated with
CAR-T therapies. They include severe and life-threatening toxicities, limited efficacy against
solid tumors, inhibition and resistance in B cell malignancies, antigen escape, limited persis-
tence, poor trafficking and tumor infiltration, and the immunosuppressive microenvironment
(Sterner and Sterner, 2021).
Before cell therapies can be tested in the clinic, nonclinical studies evaluating the effi-
cacy, distribution and potential toxicities must be conducted. This chapter focuses o n
nonclinical studies with cell therapies (with a focus on differentiated stem cell and
CAR-T therapies) and nonclinical development challenges/toxicities associated with these
modalities.
Basic principles based on regulatory guidance
Nonclinical studies with cellular therapies are conducted to: (1) determine efficacious and
safe doses, (2) identify potential target organs, and (3) provide guidance to clinicians on
selecting the patient population, starting clinical dose, and monitoring strategies for potential
10. Considerations for nonclinical studies conducted to support the development of cellular therapies316
III. Specialty biologics and indications
toxicity. Designing a nonclinical strategy to assess for efficacy and toxicity should be done on
a case-by case basis. However, when constructing a nonclinical development strategy for a
cell therapy, key elements that should also be considered include: prope rties of the test article,
the clinical indication, and the patient population. These factors can influence the types of
in vitro studies conducted, as well as critical aspects of the in vivo studies, including species
selection, use of disease models versus normal animals, route of administration, the duration
of studies, and doses to be tested. In addition, available regulatory guidance should be
considered (FDA, 2013, 2022; EMA, 2019).
The intrinsic properties of your test article such as the type of cell therapy, the source of
cells used in the cell therapy, and the use of edited cells, and/or the inclusion of functional
elements can impact the nonclinical development strategy in multiple ways.
(A) The Type of Cell Therapy (i.e., Stem Cells vs. CAR T)
Stem cells can divide and differentiate into various cell types, a process typically
controlled by the physiological environment of the host where the cells have resided
following in vivo administration. Efficacy and toxicity of stem cells are influenced by
their origin (donor and tissue sources), the stage of differentiation when administered,
and the level of manipulation of the cells. For this type of therapy, it is critical to
ensure that your population of cells are not contaminated with undifferentiated stem
cells; and it is usually necessary to remove incompletely differentiated progenitor/
precursor cells (typically with manufacturing controls). It should be noted that
although functionally differentiated cells are typically unable to proliferate and
differentiate into multiple cell types, an assessment for their ability to retain
characteristics of the cell of origin and in vivo stability (i.e., properties did not change)
in the host environment should be considered.
The potential toxicity of CAR-T cells can be influenced by the antigen recognition
domain, the target antigen, T-cell population used to express the CAR, and/or the
genetic relationship of the CAR-T cells to the patient’s cells (allogeneic vs. autologous).
Target specificity is impacted by the antigen recognition domain. Preclinical
evaluations to assess for specificity and affinity of the anti gen recognition domain
should include an evaluation of the potential for unintentional involvement of
nontargeted/normal tissue that either expresses the intended target antigen (on-
target/off-tumor), and/or other antigens. This can be evaluated by: (1) tissue cross-
reactivity studies with a monoclonal antibody (or fusion protein) that has the same
antigen recognition domain; (2) testing for cytotoxicity in cells from different tissues/
organs; (3) protein arrays; and (4) relevant animal models. Potential off-target activity
can be helpful for enrolling the correct patient population and impact the clinical
monitoring strategy. Cells typically used to express the CAR include (1) purified T cell
subsets; (2) pools of unselected T cells containing other contaminant cells (e.g., NK
cells, B cells, etc.); (3) T cells specific to viral antigens (e.g., cytomegalovirus (CMV),
EpsteineBarr virus (EBV)); and (4) selected stem-like or “young” T cells. To address
the risk for uncontrolled proliferation and toxicity, preclinical evaluations may include
examination of cytokine-independent cell growth, in vitro and in vivo testing for T cell
clonality, karyotypic analysis , TCR repertoire analysis, and specificity for viral
antigens through ex vivo stimulation and recognition assays.
Basic principles based on regulatory guidance 317
III. Specialty biologics and indications
(B) Source of Cells Used in Cell Therapy
Safety and efficacy of the test article is influenced by the source of cells that are
used. Stem cell therapies typically involve stem-cell-derived, or functionally
differentiated-derived cells. Stem cells can be sourced from adult (e.g., hematopoietic,
neural, mesenchymal, cardiac, adipose, skin); perinatal (e.g., placental, umbilical cord
blood); fetal (e.g., amniotic fluid, neural); and embryonic tissues. Functionally
differentiated cells can be sourced from various tissues (such as immune cells,
pancreatic islets, hepatocytes, neuronal cells, etc.). Understanding the source of cells
used in the cell therapy is important because evaluations for potential toxicities may
be dependent on the function of the source d cells.
Cells used in cell therapies can be autologous (cells removed from the host and
modified cells injected back into same patient) or allogeneic (a healthy donor’s cells
are injected into a patient). With allogeneic cells, there a greater risk for an immune
response. As such, evaluations for graft versus host disease or host rejection should be
considered, where appropriate, because these findings can impact the study duration
and potentially confound interpretation of results. Measurements for acute graft
versus host disease include: death, weight loss, chimerism in lymphocyte populations,
histopathology changes in the intestine, liver, or skin, increased inflammatory
cytokines (such as interferon gamma (IFN-g), tumor necrosis factor alpha (TNF- a) and
interleukin 1 beta (IL-1b)), and/or deficient T and B cell proliferation. Measurements
for chronic graft versus host disease include: T helper type 2 (TH2) clones or
cytokines, serum immunoglobulin E (IgE), lack of antihost cytotoxic T lymphocyte
(CTL), rheumatoid factor, antibodies to single strande d (ss)DNA, ribonucleoprotein,
renal tubular antigens, and laminin (Hakim et al., 2001). Evaluations for host rejection
include mixed lymphocyte reactions or HLA typing.
(C) Modifications to Cells Used in Cell Therapy
Transgene product modifications to the cell therapy can also impact nonclinical
studies and evaluations. For example, the addition of a suicide gene to the cell therapy
products can lead to selective ablation/elimination of all the gene-modified cells (and
only these cells) and prevent damage to nearby normal cells/ tissues. Suicide genes
can have mechanisms of action that are metabolic (e.g., herpes simplex virus
thymidine kinase), dimerization inducing (e.g., inducible Caspase9 (iCasp9)/AP1903)
or therap eutic monoclonal antibody (e.g., CD20 and anti-CD20 monoclonal antibody)
mediated (Jones et al., 2014). When used, nonclinical studies must demonstrate their
function and establish dosing of any additional drug or biologic needed to induce cell
elimination. Another modification to the transgene product used in cell therapy is
editing the transgene product to correct/silence an incorrectly functioning protein or
induce a correctly functioning product. In this case, assessments for off-tar get editing
of the DNA and potential integration of the vector used to deliver the transgene
product may be warranted. Gene editing techniques are also used to decrease
immunogenicity or increase persistence. In this case, studies evaluating mixed
lymphocyte reactions or cytokine-independent growth may be warranted to assess for
effects on immune function and uncontrolled proliferation, respectively. A third
modification is to include one or a combination of activation or costimulatory domains
(such as CD3z chain, 4-1BB (CD137), CD28, and CD40) into the transgene product.
10. Considerations for nonclinical studies conducted to support the development of cellular therapies318
III. Specialty biologics and indications
The addition of these domains can impact cytokine secretions, and ultimately cell
expansion, persistence, and activation of other immune cell types. To assess for this
potential toxicity, evaluations for cytokine-independent growth, and/or
histopathology evaluations for proliferation or tumorigenicity should be considered.
The clinical indication and patient population can influence the nonclinical development
strategy in many ways. For example, patients administered iPSCs derived from blood cells,
may need to be of the same blood type as the cell therapy. The patient population can also
influence the species used in nonclinical studies, the need to use diseased (vs. normal) ani-
mals, and the age of the animals evaluated.
For cellular therapies, a combined pharmacology and toxicology (pharmtox) study is often
conducted. Advantages of the pharmtox study are (1) it allows for an assessment of efficacy
and safety in disease models; (2) it may allow for evaluations at timepoints longer than stan-
dard toxicity studies; and (3) it can reduce the number of studies (and subsequent number of
animals and test article) needed to support a regulatory submission. However, be aware that
pharmtox studies can also limit the number of endpoints (because of limitations in tissue
amounts or blood volume), and importantly, one must ensure that doses are high enough
to assess for toxicity at a multiple higher than the efficacious dose (which may not be known
at the time of conducting the study).
Other important considerations when designing the nonclinical strategy for a cell therapy
include species selection, study design components (i.e., number of groups and animals, dose,
and duration), route of administration, and GLP status.
Species selection
There are multiple regulatory guidance documents that discuss species selection for
nonclinical toxicity studies. While considering a case-by-case approach, generally more
than one species may be necessary for cell therapy programs. The species selected should
be pharmacologically active, have relevant physiology and anatomy similar to humans, be
immune tolerant to the cell therapy product, allow the ability to dose the appropriate
anatomic site with a specified number of cells, and be permissive to viral infection (if a vector
is needed). The age of the animals used should be representative of target patient population.
It is also important that the species used supports administration of the test article by the clin-
ical route of administration. In some cases, a relevant species may not be available, so the use
of a biologically active surrogate/analogous cell therapy product, may be tested. However, in
this case, analogy for the ability to harvest, identify, isolate, and expand cells, cell growth ki-
netics, and cell function and viability will likely need to be evaluated. Analogous cells should
not be used to assess tumorigenicity.
In nonclinical studies, cross-species immunogenicity to a human cellular product may
require the animal model to be modified so that it becomes immune tolerant for the admin-
istered human cells. This can be accomplished by administering imm unosuppressants to
immune-competent animals, using genetically immunodeficient animals, using humanized
animals and/or administering the test article to an immune privileged site. Genetically modi-
fied (i.e., transgenic or knock-out/in) animals are often used, when appropriate. For example,
nonobese diabetic (NOD) severe combined immunodeficiency (scid) gamma (NSG) mice with
Basic principles based on regulatory guidance 319
III. Specialty biologics and indications
interleukin 2 receptor gamma (IL2rg) gene knockout and NOD-scid genetic background,
lacking mature T, B and NK cells, are recognized as a suitable immunodeficient model for
the cell xenografting. Similarly, NSG mice humanized with CD34þ cells or peripheral blood
mononuclear cells (PBMC) can be used to assess for human-relevant immune responses to the
human cell therapy administered to animals. The use of immunodeficient animals will often
allow for an assessment of long-term safety.
The use of diseased/injured animals may also be acceptable, and in some cases preferred.
For example, if a potential treatment required the cell therapy to interact with a target that is
only present in the disease state, then the use of a diseased animal model would provide
more relevant information on the efficacy and safety of the test article. It should be ackno wl-
edged that the use of disease models also has some limitations that could interfere with the
Sponsor’s ability to interpret data and/or achieve the objectives of the study. For example,
there may be a lack of historical values for background findings, the untreated control ani-
mals may have a limited lifespan because of the disease, the characteristics of the disease
in animals may not be fully reflective of what occurs in humans, or organ toxicity caused
by the disease model are similar to those observed with the test article.
Regardless of the animal species used, the nonclinical sections of regulatory submission
documents (such as Investigational New Drug (IND)/Biologic License Application (BLA)
or Clinical Trial Application (CTA)/Marketing Authorization Application (MAA)) should
include a detailed justification for the species used to assess for pharmacology and toxicity
of the test article. This justification should also include limitations of any nonstandard animal
models (when used).
Study design components
The types of studies (i.e., general toxicity, safety pharmacology, reproductive and develop-
mental toxicity, genotoxicity and carcinogenicity), included in the nonclinical strategy are
often dependent on the indication, the patient population, biodistribution of the cell therapy,
previous experience with similar molecules, and the stage of development. For example, stan-
dard 2-year carcinogenicity studies may not be needed for an oncology indication (but an
assessment of tumorigenicity may still be warranted). Reproductive and developmental
toxicity studies may not be needed for treating indications only observed in postmenopausal
women.
Although case-by-case considerations are needed when designing a study, pharmtox or
standalone toxicity studies with cell therapies typically involve single or multiple doses of
control or test article; and there are multiple groups that are each given different doses of
test article so that a doseeresponse relations hip can be determined. Howeve r, it should be
noted that determination of a dose response may be challenging for some cell therapies. Con-
trol groups can be vehicle, undifferentiated/partially differentiated cells, and/or some form
of positive control. Each group should contain enough animals to allow for a statistical eval-
uation of the desired endpoints, as well as account for any background findings. Generally, at
least 10 animals/sex/group/timepoint are used for rodents and 3 animals/sex/group/time-
point are used for nonrodent studies. Multiple necropsy timepoints may be needed based on
the mechanism of action, the need to assess for survival, engraftment and biological activity
that maintains functional stability over time, and cell fate. This will allow for an assessment
10. Considerations for nonclinical studies conducted to support the development of cellular therapies320
III. Specialty biologics and indications
for acute, chronic, and delayed-onset toxicity, and risk for tum origenicity/hyperplasia. When
possible, the doses selected should span the minimum efficacious dose and some multiple
above the maximum clinical dose. However, the high dose in the study may also be selected
based on a dose that represents the maximum number of cells that can be administered. The
dosing schedule and route of administration should be highly representative or the same as
what will be used in the clinic and should target the clinical anatomic site, if feasible in the
preclinical model.
Studies should assess standard toxicity endpoints (mortality, clinical observations, body
weights, physical examinations, food consumption/appetite, clinical pathology (serum
chemistry, hematology, coagulation, urinalysis), organ weights, macroscopic and micro-
scopic observations). Additional endpoints should be included based on the targeted cell
phenotype, the source of the cells and the extent of the ex vivo manipulation. Examples
of endpoints that should be considered include changes at the injection site, biodistribution,
immune response of the host, inflammation in target/nontarget tissues, safety pharmacology
endpoints such as effects on the CNS, respiratory and/or CV systems, and cell fate
postadministration.
Following administration, the fate of the cells could be one or more of the following: (1)
The cells could die or survive and engraft. (2) The cells could distribute to target and/or
nontarget tissues. (3) The cells could differentiate into a functioning cell or have no effect.
(4) The cells remain stable for a prolonged period of time or coul d proliferate uncontrollably,
and cause hyperplasia, dysplasia or tumors. To assess fate, cell survival, morphology and dis-
tribution, functional effects of on/off-target activity in target/nontarget tissues, extent of pro-
liferation, evidence for ectopic tissues and tumorigenicity are evaluated biochemically,
immunohistochemically and/or histologically over time.
Nonclinical toxicology studies should be conducted in compliance with good laboratory
practices (GLP), but some toxicology assessments may be conducted non-GLP. Since most
cell therapies, are typically long acting, many if not most of the nonclinical studies are
completed prior to first-in-human trials. However, if there are major manufacturing or
formulation changes later in development, bridging comparability studies will likely be
needed.
Development challenges
Challenges associated with stem cell therapies
When considering the development of stem cell therapies, risks for tumorigenicity, inap-
propriate stem cell migration, incorrect or unstable stem cell fate, and immune rejection of
the stem cell transplant are the development challenges of greatest concern. These potential
risks should be evaluated in nonclinical studies, and de-risked as much as possible.
Tumorigenicity
One of the most significant concerns for hESCs and iPSC s is the risk for tumorigenicity;
because stem cells have the potential to proliferate indefinitely while maintaining cell identity
(i.e., they are self-renewing), and because they differentiate into all the cell types of the three
Development challenges 321
III. Specialty biologics and indications
germ layers (i.e., they are pluripotent). The risk for tumorigenicity is further increased
because of several other factors: (1) Transcription factors that were commonly used to repro-
gram somatic cells into iPSCs (such as OCT4, SOX2, MYC and krupple-like factor 4 (KLF4))
are also commonly overexpressed in various tumors (Ben-David and Benvenisty, 2011). (2)
Some culturing processes can cause the accumulation of genetic mutations, that predispose
differentiated progeny cells to form tumors, in vivo (Werbowetski-Ogilvie et al., 2012). (3)
Stem cells may be designed to be hypoimmune (Deuse et al., 2019) and if they become
cancerous after transplantation, may not be removed by the host’s immune system. Addi-
tional evidence to support that stem cells can cause tumors/teratomas has been observed
both nonclinically and clinically. When human embryonic stem cells were injected into
immunodeficient mice, teratomas were observed. These teratomas occurred when hESCs
were administered by subcutaneous, intratesticular, or intramuscular injection, or implanta-
tion under the kidney capsule; and subcutaneous teratoma formation was increased by coin-
jecting the hESCs with a cell culture m atrix, such as Matrigel (Prokhorova et al., 2009). In
addition, human ESC-derived dopaminergic neurons and neural progenitor cells were asso-
ciated with neural overgrowths and tumors when transplanted into small animals or mon-
keys; and ocular tumors formed in mice administered ESC-derived retinal progenitor cells
(Roy et al., 2006; Kriks et al., 2011; Cui et al., 2013; Doi et al., 2012). Furthermore, a neoplasm
with glial differentiation was detected in a stroke patient with residual deficits who was intra-
thecally administered infusions of mesenchymal, embryonic, and fetal neural stem cells (Ber-
kowitz et al., 2016). More recently, acute myeloid leukemia (AML) was observed in two
patients treated with Lovo-cel, a modified hematopoietic stem cell therapy for the treatment
of sickle cell disease. However, AML was attributed to either the busulfan preconditioning, or
was deemed unlikely related to insertional oncogenesis (Kanter et al., 2023).
The risk for tumorigenicity can be assessed both in vitro and in vivo. In vitro assays can
detect residual undifferentiated pluripotent stem cells, unintended immortalized or trans-
formed cells, or genomic instability (Table 10.2). These assays are generally easy to conduct,
TABLE 10.2 In vitro methods for evaluating for tumorigenicity and their advantages and disadvantages.
Purpose Methodology Measurement Advantages Disadvantages
Detection of
pluripotent stem
cells
Flow cytometry Immunophenotyping
pluripotent stem cells
• Rapid (w1 day)
• More sensitive than
colony formation
assay
• Can distinguish,
separate, and collect
individual cells
• Detects only cells
expressing known
markers
• Gating techniques
strongly in fluence
results
Quantitative RT-
PCR, droplet digital
PCR
Gene markers for
pluripotent stem cells
• Simple and rapid
(approximately 6 h)
• Limited to detection
of cells expressing
known gene
markers
Highly efficient
culture of
pluripotent stem
cells
Colony formation of
pluripotent stem cells
• Simple and sensitive • Time consuming
10. Considerations for nonclinical studies conducted to support the development of cellular therapies322
III. Specialty biologics and indications
are rapid, sensitive and/or specific. However, they all have their limits. Pluripotent stem cells
can be detected by a variety of different methods, which include: (1) evaluating specific
protein markers by flow cytometry; (2) evaluating gene markers by quantitative RT-PCR
or digital droplet PCR; (3) culturing stem cells and evaluating pluripotent cell colony forma-
tion or specific markers released into the culture medium. However, the sensitivity and
TABLE 10.2 In vitro methods for evaluating for tumorigenicity and their advantages and
disadvantages.dcont’d
Purpose Methodology Measurement Advantages Disadvantages
Detection of marker
molecules released
into culture medium
Marker molecules for
pluripotent stem cells
• Simple and
noninvasive
monitoring of
pluripotent stem
cells
• Results influenced
by components of
culture medium and
other cell culture
conditions
Detection of
immortalized cells
Cell proliferation
assay (after serial
passage)
Cell proliferation rate • Simple and
inexpensive
• More sensitive than
nude mice
• Time consuming
Detection of
anchorage-
independent cell
growth (colony
forming capacity in
semisolid media)
Digital soft agar
colony formation
assay
Colony formation of
malignant transformed
cells
• Rapid (a few weeks
to 1 month)
• Highly sensitive,
compared with
conventional soft
agar colony
formation assay,
• Assay cannot be
applied to floating
cells
• High content
imaging system
needed
• Cannot detect
hPSCs because of
dissociation-induced
apoptosis
Detection of
genomic instability
Karyotype analysis Number, size, and
structure of
chromosomes
• Technically
established for
genotoxicity
assessment of
chemicals
• Correlation between
genetic aberration
and tumorigenicity
is obscure
• Assays cannot
detect trace amounts
of tumorigenic
cellular impurities in
hCTPs
aCGH Genomic copy number
variations
FISH assay Localization and
quantitation of specific
DNA sequences on
chromosomes
Genome sequencing
by NGS
Genomic single
nucleotide variants and
copy number variations
• NGS enables
extensive analysis of
genomic
abnormalities
aCGH, array comparative genomic hybridization; FISH, fluorescent in situ hybridization; hCTP, human cell therapy products;
hPSC, human pluripotent stem cells.
Table from Sato et al. (2019).
Development challenges 323
III. Specialty biologics and indications
specificity of these assays are dependent on the cells expressing the markers being measured,
and the culturing model can take longer and be influenced by factors in the media. Immor-
talized cells are detected by evaluating the cell proliferation rate after serial passes in the cell
proliferation assay. However, it can take some time to find contaminating cells that are pre-
sent in relatively low amounts. Colony formation of malignant transformed cells in digital
soft agar can be used to detect for anchorage independent cell growth. Although this method
is highly sensitive, it is limited by the need for a high content imaging system, which may not
always be available in certain settings. Genomic instability can be determined by karyotyping
the number and size of each chromosome; evaluating genomic copy variations (via array
comparative genomic hybridization) or specific DNA sequence on chromosomes (using fluo-
rescent in situ hybridization); or measuring single nucleotide or copy number variants in the
genome (using next generation sequencing). Unfortunately, evaluating genomic instability is
limited by the extent of the known correlation between the genetic aberration and tumorige-
nicity; and may not be sensitive enough to detect effects caused by a trace number of cellular
impurities (Sato et al., 2019).
The risk for tumorigenicity can also be evaluated in vivo. Generally, an evaluation for
tumorigenicity can be conducted in an immune deficient animal such as the NOD/SCID/
gCnull (NOG) mice (Ito et al., 2002) and NOD/SCID/IL-2rgKO(NSG) mice (Ishikawa
et al., 2005), or athymic nude rats (Kanemura et al., 2014). When considering which species
to select, factors such as dose, and route of administration can influence the need for larger
animals (such as the athymic nude rat) over mice. Regardless of which model is selected,
proof of engraftment and survival of the transplanted cells must be demonstrated for the
assay to be valid. However, one must also consider that the microenvironment of the stem
cells (where tumorigenicity is being assessed) is not entirely representative of that of patients.
For example, the engrafted and potentially tumorigenic cells in the NSG mouse model only
remain engrafted because the immune system is absent in NSG mice (which will not be the
case in patients). However, NSG mice engrafted with CD34þ human hematopoietic stem
cells (hu-CD34þ HSCs) and have functional CD4þ and CD8þ T cells, macrophages, den-
dritic, and NK cells (Ishikawa et al., 2005), were not more sensitive than the NSG immuno-
deficient model for studying the malignancy of stem cells (Bialecka et al., 2022). Lastly, the
in vivo study must test at least the same dose of cells administered to patients, or the
maximum feasible dose that can be adm inistered to the test species, or a dose that has
been scaled down (based on size, organ weight or volume) to represent the clinical dose in
the smaller animal species.
The risk for tumorigenicity may be further mitigated by engineering/genome editing cells,
to manipulate genes involved in tumor formation, or to introduce safety switches into human
iPSCs, so that it allows for a small molecule to selectively kill undifferentiated human iPSCs
or to efficiently eliminate the entire cell product, if necessary. For example, Martin et al. (2020)
demonstrated that preci sely editing in NANOG
iCasp9
into the endogenous loci of human
iPSCs enables the small molecule, AP20187, to kill undifferentiated teratoma-forming cells
from a therapeutic human iPSC-derived cell product, prior to transplantation. Similarly,
engineering human iPSC to inclu de ACTB
TK
or ACTB
OiCaspase9
allows the small molecules
ganciclovir or AP21967, respectively, to rapidly eliminate the entire cell product (not just
the plur ipotent cells) if an adverse event was to occur (Martin et al., 2020).
10. Considerations for nonclinical studies conducted to support the development of cellular therapies324
III. Specialty biologics and indications
Inappropriate stem cell migration
Inappropriate stem cell migration is another potential risk. In a publication by Master et al.
(2007) they discuss how patients receiving stem cell transplants were at risk for developing
seizure-like symptoms or possibly brain dysfunction that are caused by stem cell migration
from the graft site to inappropriate regions of the brain. Understanding where the cell ther-
apy distributes to and quantifying how much is there, is imperative for estimating the safety
of cell therapies. The ability for cells to distribute to unintended tissues can be evaluated non-
clinically by collecting different tissues and measuring for the presence of a human gene/
gene product in various tissues. This can be accomplished using quantitative PCR measure-
ments. Alternatively, if cells were constructed to express luciferase, in vivo bioluminescence
imaging of the luciferase expressing cells can also be used to evaluate for inappropriate
migration. When conducting these assessments, multiple timepoints are typically needed
and one should ensure that the study was of sufficient duration to be able to evaluate distri-
bution (or re-distribution) to various tissues over time. The risk of stem cell migration can be
reduced by injecting to the desired site (vs. systemically), confining the stem cell to within an
implantable device, or by injecting fully differentiated cells.
Cell fate
Prior to administration, cell fate is evaluated to ensure that the stem cells differentiated
into the target cell type, and that stem cells do not differentiate into an unintended cell
type over time. This can be accomplished by demonstrating appropriate (1) cell morphology,
(2) lineage specific markers, (3) gene expression, and (4) cell function that remains stable over
time (Nguyen et al., 2010). Cell morphology can be evaluated microscopically. However, cell
morphology alone, cannot be the only parameter used to confirm cell fate because the cells
must also be functional to be therapeutic. Lineage specific markers such as cell surface recep-
tors or intracellular proteins can be used to confirm the stem cell has differentiated into a spe-
cific cell type. Examples include cardiac markers such as GATA-4 or atrial natriuretic peptide;
nervous system markers such as CD133, O1, O4 or glial fibrillary acid protein; or pancreatic
cell makers for cytokeratin 19, insulin or glucagon. Generally, an antibody/signaling mole-
cule containing a fluorescent tag that has strong affinity for the lineage marker is added to
the cells, the cells are sorted by FACS, and then identified using immunofluorescent or immu-
nohistochemical staining. Similarly, unique patterns of gene expression can be detected by
reporter genes, quantitative real time polymerase chain reaction (RT-PCR), and microarray
analysis. Examples include upregulation of IsL1, or Nsg2, in cardiac, or neuronal stem cells,
respectively. Lastly, cell function can be evaluated by a variety of different methods depend-
ing on cell type. This can include patch clamp responses of different ion channels, that can
correlate to biological functions such as spontaneous contractions of cardiomyocytes or action
potentials of neurons.
Immune rejection
As mentioned previously, stem cells administered to patients can be autologous or alloge-
neic. Autologous stem cells are collected from, and administered back into, the same patient.
For example, a patient’s hematopoietic stem cells are isolated, stored, and following
chemotherapy, are administered back to the same patient to replace those cells destroyed
Development challenges 325
III. Specialty biologics and indications
Соседние файлы в папке Библиотека им академика М.И. Перельмана