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Determining a threshold for residual stem cells
Although healthy, properly derived PSCs should not carry any oncogenic mutations and
would be screened for such, as described above, the concept that cancer stem cell compart-
ments give rise to malignant cancers suggests a related phenomenon of teratoma growth if
compartments of trace residual PSCs were retained in the differentiated product. However,
even within the human cancer stem cell field, few assays can reproducibly predict the
threshold for tumor formation. While it is clear that many factors can influence tumor or tera-
toma formation, perhaps the strongest driver is the genomic stability of the cellular product.
While the starting cell source material (iPSC vs. ESC, specific cell line used, specific cell bank
used) might affect teratoma formation potential (Hong et al., 2013), as well as the propensity
for resi dual stem cells to persist through the manufacturing process, it is unknown to what
degree these “ cell-autonomous” factors exist within PSC populations and influence the
threshold to form a teratoma. In fact, even within the PSC field, discrepancies remain in
the reported threshold number of cells required for teratoma formation in mice, varying
from as few as 2 to as many as 100,000 cells (Hentze et al., 2009; Lee et al., 2009; Shih
et al., 2007; Lawrenz et al., 2004; Cunningham et al., 2012). The challenge for sponsors is to
TABLE 12.8 Product characterization.
Assessment Evaluation criteria
Identity Presence or absence of phenotype-specific markers
Protein secretion
mRNA and microRNA expression
Morphology
Adherence
Differentiation potential
Purity Absence of cell contaminant markers
Marker expression
Cellular and noncellular impurities profile assessment
Potency Expression or secretion of relevant biological substances, such as recombinant proteins,
glycoproteins, lipoproteins, growth factors, enzymes, cytokines, etc.
Formation of cellular and extracellular matrices
Formation of cellular and extracellular structures
Cell-to-cell interactions
Migration
Proliferation or differentiation
Stability Proliferation
Recovery
Stress markers
Phenotypic stability
Safety Absence (or below tumor formation threshold) of residual PSCs within product
Stable growth kinetics
Chromosomal stability
Other MHC/HLA expression
12. Considerations in the development of pluripotent stem cellebased therapies416
III. Specialty biologics and indications
define the series of assays that will provide confidence that any residual stem cells that might
remain in the population are below the threshold that would result in teratoma formation
(Sato et al., 2019), since even if the growth is benign, it could still be problematic.
Accordingly, sensitive assays are emerging that can aid in determining if undiffere ntiated
PSCs exist within the final cell product (Cu nningham et al., 2012; Kuroda et al., 2015; Bagh-
baderani et al., 2015). Flow cytometry-based assays require the use of markers that can reli-
ably identify the presence of undifferentiated or partially differentiated PSCs; molecular
assays such as quantitative polymerase chain reaction (qPCR) and digital drop PCR (ddPCR)
provide sensitive measures of gene expression and are becoming more prevalent methods of
characterizing product purity for release prior to administration. Sponsors will also need to
estimate the proportion of the clinical product to be sampled to assure statistical certainty that
there is no or very low potential for teratoma/tumor formation. Knowing the cell dose to be
administered in patients can aid in determining what portion of the product will need to be
tested to provide confidence, as well as helping to guide the design and pow er of any in vivo
tumorigenicity studies.
Kinetics, chromosomal stability, differentiation status, and cellular attributes
Most spontaneously occurring tumors are marked by having unstable/accelerated growth
kinetics, chromosomal aberrations, dysregulation of key tumor suppressors or oncogenes,
presence of tumor-forming units, and or the appearance of immature/undifferentiated cells.
It may be beneficial to demonstrate that prior to differentiation the PSCs lack these features
and instead exhibit characteristics of “normal” diploid somatic cells that carry little or no risk
for tumor formation.
Growth kinetics of the starting population can be determined by measuring cell doubling
time, proliferation rates, or cell cycle kinetics by flow cytometry. Whichever assay is used
measurements should be made over multiple cell passages in order to demonstrate balanced
growth typical of “normal” PSCs.
Chromosomal stability (e.g., G-banding, etc.) provides information about the integrity
and functionality of cellular repair, replication and checkpoint controls. Standard karyotyp-
ing involves assessing dividing cells in metaphase. Certain karyotypic aneuploidies are
recurrent in PSC cultures, especially if extended passaging is required; for example, a gain
of chromosome 20q11.21, which includes the BCL2L1 gene, is not uncom mon (Wu et al.,
2008). Mutant cells overexpressing Bcl-xL, the predominantly expressed isoform of
BCL2L1, have a selective advantage in culture, and can also be impaired in their differenti-
ation capacity (Markouli et al., 2019). Qualified cytogenetic laboratories can assist in this
interpretation and sponsors can refer to the principles in the International System for Human
Cytogenetic Nomenclature (Shaffer, 2013). More recent advances in chromosomal integrity
assessment include chromosome microarrays, optical genome mapping, and other methods,
which have improved sensitivity and automation over traditional cytogenetics met hods
(Keller and Spits, 2021; Kitano et al., 2022). Additionally, molecular genetic analyses that
look for sequence modifications such as single nucleotide variants (SNV), dinucleotide var-
iants (DNV), inse rtions, deletions, and copy number variations (CNV), may be useful to
identify specific undesirable sequences of particular interest for safety, such as mutations
in TP53, BCOR, HER2,orJAG2 ( Merkle et al., 2017; Rouhani et al., 2022; Yamamoto et al.,
2022; D’Antonio et al., 2018). Irrespective of the methods used, sufficient sampling must
Important early decisions: Chemistry, manufacturing, and controls 417
III. Specialty biologics and indications
be applied to ensure statistical confidence in extrapolating that the cell population is stable
and free of chromosomal abnormalities.
Differentiation status of the cell product can be determined by using specific markers of
pluripotent, progenitor, and differentiated cell populations. Cell surface and intracellular
antibody markers can be used to identify cells by immunocytochemical or immunohisto-
chemical labeling, or flow cytometry. Quantitative RT-PCR, immuno-PCR, ddPCR, and sin-
gle cell RNAseq are alternative methods that can also be used to estimate the various cellular
fractions within the cell product to infer differentiation status and cellular attributes.
Potency assays
Phenotypic and morphological assays alone are generally not sufficient predictors of the
biological activity of a cell product; therefore, efforts should begin early to develop a potency
assay. Potency is defined as the specific ability or capacity of the product to effect a given
result. In other words, it is the strength of the therapeutic activity of a product as determined
by quantitative measure of relevant biologic function of the product based on the attributes
that are linked to its rele vant biologic properties (Bravery et al., 2013; FDA, 2021). A potency
assay provides a mechanism to assess the quality, strength, consistency, and stability of a
product throughout all stages of clinical study. In addition, it can be used to assess compa-
rability of the product during changes in manufacturing that may occur such as scale-up,
site change, process improvements, transition to new starting materials (cell line), or changes
in critical reagents or formulation. This is partic ularly important with PSC-based therapies as,
without a way to assess lot-to-lot comparability, even minor process change s could require
costly and time-consuming qualification studies. Therefore, developing potency assays early
on will not only ensure the consistency and strength of a product to bring about desired clin-
ical effect, but also provide a tool for establishing product comparability and enable process
changes in the future (Bravery et al., 2013).
The design of the potency assay will depend upon the nature of the specific product and
often requires an understanding of the underlying mechanism of action. Examples of biolog-
ical activit y include expression of key proteins (including growth factors, enzymes, or cyto-
kines), formation of cellular or extracellular structures, and up- or dow n-regulation of
inflammatory or immunological activity (EMA, 2011 ). A matrix approach to potency should
be implemented using a combination of different types of assays, such as functional tests,
marker-based assays, and both in vitro and in vivo assays to assess the biological activity
of the product (Bravery et al., 2013). Because ef ficacy is considered to be proportional to
the potency of a product, the goal of such assays is to show a correlation with the intended
therapeutic effect in a quantitative or semiquantitative manner, if possible. Potency assess-
ment will likely evolve during the product development proces s as new information is
learned from in vitro and in vivo nonclinical studies, and early clinical studies. Assays
used for release of product lots used for early clinical studies are likely to have wider accep-
tance criteria than assays used in later phase investigations, as release criteria are likely to
narrow with additional characterization experience. While process improvement of the po-
tency assay can continue during development, is expected that sponsors provide a validated
potency assay or assay matrix at pivotal clinical testing.
12. Considerations in the development of pluripotent stem cellebased therapies418
III. Specialty biologics and indications
Product lot release spe cifications
Specifications for release of a cell product should be based on characterization elements
discussed above, as well as additional quality metrics such as stability, viability, and safety.
Parameters should be established for identity, purity, and potency based on data obtained
from rigorous in vitro and in vivo testing of the product and should be optimized in an iter-
ative process throughout early product development.
The manufacturing of PSC-based products typically occurs over the span of several weeks
to months and involves the extensive manipulation of cells during culturing. Like many bi-
ologicals, live cell-based products cannot undergo terminal sterilization and therefore must
be manufactured under aseptic conditions. Extensive testing must be performed prior to
administration to patients to ensure that the product is free from microbiological contamina-
tion through sterility, mycoplasma and endotoxin testing (Rayment and Williams, 2010). In
addition, since many of these products must be administered to the patient within a short
time from final product formulation (if not cryopreserved), results of sterility testing may
not be available prior to administration; in that case rapid and full release wi ll need to be con-
ducted, consisting of an in-process sterility test showing no-growth of a sample taken at
48e72 h prior to harvest plus negative gram staining on the final formulated product and
14-day sterility testing in parallel. It is required that sponsors developing products released
prior to the availability of 14-day sterility test data fully describe the procedures followed
in the event of sterility failure; 21 CFR 312.31 prescribes reporting sterility failure, root cause
investigation and corrective actions within 30 calendar days after initial receipt of the positive
culture test result. If the product will be cryopreserved prior to use, release-testing for micro-
biological contamination should be performed on product prefreeze and postthaw, as part of
a stability program.
The effects of a cell-based product typically depend on the presence of viable cells. There-
fore, it is essential to establish minimum viability requirements for lot release. Several
methods and instruments exist for cell counting and viability assessment, and it is adv isable
to determine which might be most accurate and useful for the product in development. In
addition, specifications for cell concentration should be defined, as density and cell dose
likely affect product activity. It is prudent to develop release specifications prior to start of
pivotal nonclinical studies, as the product used to assess nonclinical safety should be compa-
rable with clinical lots.
Nonclinical development
Principles of nonclinical development
The purpose of the nonclinical section of a regulatory submission is to describe in detail the
pharmacologic and safety pro files of the cell therapy to support a safe starting dose for testing
in patients. The data package typically includes: a description of the product to be tested and
the intended patient population, justification of the models used to evaluate mechanism of
action and safety, and a clear and cogent description of the in vitro and in vivo studies
that were conducted to understand the pharmacology, pharmacokinetics/biodistribution,
and nonclinical safety of the product. The nonclinical summary should then describe the
Nonclinical development 419
III. Specialty biologics and indications
key data and dose equivalence calculations that link the minimum efficacious dose (MED)
determined from pharmacology studies with a projected safe starting dose for humans.
This dose calculation should be inclusive of the safety margin relative to the no-observed-
adverse-effect-level (NOAEL) achieved in the toxicology study. Finally, when possible, trans-
latable biomarkers should be identified in nonclinical studies that can be used to monitor
either on-tar get efficacy or safety in patients in the clinic. This section of the chapter will focus
on the pharmacologic, pharmacokinetic/biodistribution and safety assessments that may
form part of the package supporting a typical PSC-derived therapeutic.
PSC-based therapies differ from small molecule drugs in that they generally cannot be
removed or have their effect reversed, making testing in normal healthy subjects in a tradi-
tional Phase 1 safety trial ethically questionable. While some encapsulated products offer
added safety because they can be removed, Phase 1 testing of these products is still likely
to occur in a diseased patient population. While the primary focus of first-in-human trials
is safety, when studies are conducted in subjects with disease, there can also be an expecta-
tion that the product might modify disease progression or even lead to functional improve-
ment. In particular, regulatory authorities have an expectation that treatment of pediatric
populations with novel cell and gene therapies includes a justification that the initial clinical
starting dose may provide some benefit. This places a greater burden on the early nonclinical
development process for novel cell-based therapies to demonstrate pharmacologic efficacy in
animal models of human disease prior to clinical entry. Thus, careful attention must be
applied to selection of an appropriate model(s), choice of endpoint measures, reproducibility,
and understanding of model limitations. Developing a robust nonclinical package requires
thoughtful development and, when possible, engaging in an early dialog with the respective
regulatory authority.
Selection of test article for nonclinical studies and key manufacturing
considerations
Testing of human PSC-derived products in animal models necessitates the use of either
immunocompromised or immunosuppressed animal models. The xeno-rejection of the hu-
man cells by the animals’ immune system is a barrier to successful product testing without
the use of genetically modified animals. To date, this has held true even for hypo-immune
cells that have been edited with the intention to “cloak” the cells from the human immune
system; the xeno-engraftment barrier is very strong. An alternative approach for nonclinical
testing is to use an analogous PSC-derived cell product from the animal for testing in the
same species, thereby effectively making it an allo- or auto- (as opposed to xeno-) transplant.
However, investigators should engage with the regulatory agency at an early stage of prod-
uct development if they are considering making their nonclinical package dependent on an
analogous cell product. Key differences in the manufacturing of the analogous cells may
not adequately represent the safety risks of the human PSC-derived product, and in general,
results from an analogous product are viewed as less relevant because (by definition) the
tested cells are not the product candidate that will be investigated in the human clinical trials.
If an edited cell product is to be used, special consideration will need to be given to the
safety risk assessment performed on the reagents used in the manufacturing process and
12. Considerations in the development of pluripotent stem cellebased therapies420
III. Specialty biologics and indications
the genomic stability of the cell product (both before and after editing). This is discussed
above in the section on manufacturing.
It is also important to demonstrate that product cells are stable in their storage media, the
intended administration vehicle, at various temperatures, and when exposed to other rele-
vant conditions, such as those that could be experienced during shipping or after dose prep-
aration for clinical delivery. This stability testing will define the “shelf life” of the product
from formulation to clinical (and nonclinical) administration and should be part of the
CMC assessments that support the nonclinical good laboratory practice (GLP) safety/toxicity
studies.
Justification for route and method of administration for nonclinical studies
The primary reason for justifying the route of administration is to ensure that the design of
the nonclinical studies supports the intended clinical route. Secondarily, the justification
should support an understanding of delivery to the intended target site and any potential ef-
fects on that target. For many PSC-based therapies, the target site is usually at or near the site
of cell loss or injury (e.g., degenerated heart tissue, injured spinal cord), and the route of
administration mimics the clinical route (intracardiac, intraspinal). Therapies that aim to
replace a secreted factor with a systemic effect (e.g., insulin, thyroid hormone, parathyroid
hormone, etc.) are exceptions, as cells can be transplanted into distal sites, with or without
encapsulation. In these cases, efficacy studies will need to demonstrate that the animal model
contains a comparable target for the secreted human factor to effect change and that the route
of administration can accurately deliver the product to this target.
Method of administration is also critical to justify and may require additional studies to
evaluate local tolerance for the intended target, together with an assessment on the survival
and stability of the cellular product. Cells can be delivered using a syringe via catheter or nee-
dle, manually or with an automated system, with a custom device, and/or potentially on a
scaffold. In all cases, it is critical to perform (a) biocompatibility of the delivery system and
(b) dose assurance studies to confirm the number and viability of the cells being administered
are as intended. Cells can pote ntially be sheared or adsorbed, and may clump or settle, lead-
ing to various unintended effects on dosing, depending upon the method or device used for
administration. Because cells tend to adhere to various materials, it would be possible to un-
intentionally administer dead or damaged cells, or unreliable quan tities of cells. Dose assur-
ance can be performed as a series of in vitro studies in the laboratory utilizing the exact
delivery system, rate, temperature, time, and vehicle to be used in clinical administration.
Further stringency can be applied by performing postdosing analysis to determine cell
viability 24 or 48 h after “mock” administration. This accounts for the possibility that, while
cells can pass a simple viability test, they might be unhealthy or internally slated for cell death
via apoptotic or other pathways. Lastly, for more complex delivery devices including cathe-
ters that may have to be mounted to a frame or traverse an extensive part of the vascular sys-
tem to reach their target, direct in vivo testing of the clinical device may be necessary. In these
instances, large animals will likely be necessary and studies will have to incorporate a care-
fully developed immunosuppression protocol to afford successful engraftment of the cell
product. If a novel device is used to administer, deliver, or encapsulate the PSC-based ther-
apeutic, US review will require additional testing in accordance with the Center for Devices
Nonclinical development 421
III. Specialty biologics and indications
and Radiological Health (CDRH) to assure safety and biocompatibility of the materials used.
This is to assure safety of the materials to which patients will be exposed and to demonstrate
that the component delivery materials do not affect survival or activity of the cellular thera-
peutic. An understanding of the goal of clinical testing should drive the selection of the
methods used in the animal studies, so that cells are examined under conditions that reflect
the clini cal scenario as closely as possible.
Selection of animal model(s) and/or species for nonclinical studies
Typically, pharmacologic studies are conducted in an animal model of human disease,
whereas toxicology studies tend to be conducted in healthy animals. Uniquely, for cell and
gene therapies the regulatory authorities: (1) encourage the use of animal models of disease
for what are sometimes referred to as Hybrid Pharmacology Toxicology studies, where
possible, and (2) do not require the use of two species to ide ntify the most sensitive toxicology
species. The choice of an appropriate animal model will need to consider the following: pres-
ence of the pharmacologic target in the animal, availability of quantitative endpoints for
assessing efficacy, model limitations, robustness, and reproducibility. Animals used in
nonclinical studies need to be immunocompromised through genetics, ablation, or chemicals
due to the xenogeneic immune barrier (Table 12.9). Benefits and limitations of each of these
models are discussed below.
TABLE 12.9 Classes of immunologically modified animal models.
Immunocompromised Immuno-ablated Immunosuppressed
Model origin Spontaneous mutant
Transgenic knockout
Thymus removal
(thymectomy)
Gamma irradiation
Drug-induced (glucocorticoids,
antiimmunophilins, antibodies,
antimetabolites, etc.)
Immune cells/
pathway affected
T cell, NK cell, or T, B, and
NK cells
T Cell only, or T, B, and NK
cells
T cell, cytokines, or chemokines
Species tested Mice, rats Mice, Guinea pigs, pigs Mice, rats, pigs, nonhuman
primates
Pros Easy to handle
Large “n”s can be tested
Can use with lesion/toxin
models
Can be used in multiple
species
Can use with lesion/toxin
models
Can be used in small and large
animals
Can use with lesion/toxin models
Cons Small size limits cell dose
Residual immunity
Spontaneous tumors
Irradiated animals are short
lived
Need for special equipment
Immunosuppression is short lived
opportunistic infections and
tumors
Leukopenia
Autoantibodies to suppressants
Development
questions best
addressed
Pharmacology
Tumorigenicity
Toxicity
Pharmacology Site tolerance
Dose tolerance
Surgical feasibility
12. Considerations in the development of pluripotent stem cellebased therapies422
III. Specialty biologics and indications
Genetically immunocompromised models
Rodent species are attractive for proof of concept (POC) testing in early discovery.
Because of ease of handling and husbandry, large group sizes can be used to support sta-
tistical confidence, a wide range of genetic, lesion/toxin models of human disease are
available, and an array of gene and protein tools have been developed to facilitate
endpoint analysis. The most commonly employed immunocompromised models are in
mice (Chen et al., 2022). Athymic/nude mice carry a mutation in Foxn1 gene and thereby
lack mature T-cells, while severe combined immune-deficient (SCID) strains of mice and
rats carry a mutation of the Prdkc gene and lack T and B cells but retain natural killer
cells (NK) and macrophages. Rag1/Rag2 null mice also lack mature T and B cells similar
to SCID mice and are reportedly less “immunologically leaky” than SCID mice (Chen
et al., 2012). Nonobese diabetic (NOD) mice have defects in their innate immune system,
with low NK and macrophage activity and no detectable circulating complement (Chen
et al., 2012). The backcrossing of IL-2 receptor gamma null mice onto NOD and
SCID backgrounds (Ito et al., 2002) has produced newer strains (e.g., NOG, NSG,
NRG, and BALBc/Rag2/IL-2 mice) that reportedly overcome the “immunological leaki-
ness” and spontaneous tumors apparent in other immune-deficient mice, increasing life
span and overall health. More recently, additional modifications have given rise to strains
including NSGB2m, NSG-SGM3, BRGS, and DRAG mice (Adigbli et al., 2020). These mice
either lack MHC class I (beta 2 microglobulin); express human IL-3, GM-CSF, and SCF;
express the NOD-type SIRPalpha; or express HLA-DR0401 molecules respectively (see
Table in (Adigbli et al., 2020) for a summary). These recently established immunocompro-
mised humanized strains of mice offer stem cell scientists a broader range of immune-
deficient models that have improved xeno-engraftment success. Because human bone
marrow, thymocytes, and other hematopoietic/immune cells can be more effectively
engrafted into these mice, they can be used nonclinically to predict the potential inter ac-
tion of a human immune system with the product. Several experiments have used these
models to evaluate human immune-mediated rejection of human beta islet cells, vascular-
ized skin, artery segments, and more (Macchiarini et al., 2005; Shultz et al., 2007; Chen
et al., 2022). Investigators should be aware that some of the newer strains are fragile,
requiring additional husbandry and care for their successful use.
While immunodeficient mouse models are more common than rat models due to the ease
of genetic manipulation, the recent development and commercial distribution of the seve rely
immunodeficient SRG rat (Noto et al., 2020) has provided an alternative rodent model for
nonclinical studies. Rats have a size advantage over mice (10-fold larger) that may better sup-
port dose and ROA considerations. Furt hermore, with the advent of genome editing, the gen-
eration of immunodeficient large animals is now a reality (Sato et al., 2016; Maynard et al.,
2021). While not commercially available, pigs and nonhuman primates carrying SCID, IL-2
receptor gamma and/or Rag1/Rag2 mutations have been reported, which may provide addi-
tional development models (see (Adigbli et al., 2020) for a review). Importantly, there has
been no specific regulatory requirement to use nonrodent species (FDA, 2013). However, spe-
cific concerns, including tolerability testing of clinical scale dose/delivery device, might
compel testing in larger animals.
Nonclinical development 423
III. Specialty biologics and indications
Immuno-ablation models
Immune ablation requires either remova l of the thymus (thymect omy) early in develop-
ment to halt T-cell maturation, or intensive gamma irradiation to destroy bone marrowe
derived B cells. These techniques are not commonly used due to the short lifespan of these
animals once the thymus is removed or animals irradiated, but offer alternative strategies
to access models not supported by mice or rats (e.g., guinea pig models of ventricular
arrhythmia or asthma).
Combinatorial models can also be generated that include use of transgenic immunodefi-
cient animals (NRG, NSG, and NOG) with sublethal irradiation techniques followed by trans-
plantation of human peripheral blood mononuclear cells or CD34
þ
hemopoietic stem cells to
allow for generation of mice with a human immune system (HIS) or cotransplantation of hu-
man bone marrow/liver/thymus (BLT) (Walsh et al., 2017; Allen et al., 2019; Stripecke et al.,
2020; Flahou et al., 2021; Kitsera et al., 2023). The specific methods used to generate these an-
imals does vary widely between laboratories so investigators should carefully consider their
goals. Consortia exist that have attempted to establish standards for the generation of human-
ized mice and may provide helpful tools (Stripecke et al., 2020). Similar to animals who have
received immune ablation only, HIS mice have a limited lifespan and with time do succumb
to graft versus host disease (GVHD), so their utility is limited to acute timepoints in
assessments.
Chemical immunosuppression models
Immunosuppressive regimens can be used as an adjunct in immunocompromised animals
to improve engraftment, to test impact of a potential immunosuppressant on the cell therapy,
or to allow testing in immunocompetent large animals. When used in large animals, multiple
immunosuppressive drugs may be necessary to block both innate and adaptive immune re-
sponses (Thompson et al., 2012; Demkes et al., 2021). This strategy of combinatorial immuno-
suppressants has improved outcomes in patients undergoing organ transplantations (Posselt
et al., 2010). However, long-term immunosuppre ssion can lead to complications such as
opportunistic infections, injection site reactions from multidose administration, and most
significantly, neutralizing anti-Ig responses that lead to transplant rejection. This generally
means that only acute/short-term studies can be performed with immunosuppression.
Emerging is the possibility of combining novel immune therapies with human PSC transplan-
tation in large animals (e.g., T-regs or CTLA4 Ig gene therapy with immunosuppressants).
Several landmark papers have shown short term engraftment success for up to 3 months
in nonhuman primates and pigs (Chong et al., 2014; Liu et al., 2018; Romagnuolo et al.,
2019). This is a promising field that is already demonstrating results for allograft organ trans-
plantation (Hayashi et al., 2000, 2005; Demkes et al., 2021).
The emergence of new models such as humanized mice and transgenic immunodeficient
pig strains created through gene editing (Wolf et al., 2019), the application of combinatorial
immunosuppressants, and the development of novel immune therapies, collectively offer a
wide variety of opportunities for nonclinical testing. The discussion is beyond the scope of
the present chapter but it should be noted that an entire field of in vitro toxicity testing,
particularly enabled by the arrival of human PSC-derived tissues and systems, and thus
directly pertinent to human reactivity, is emerging and may gradually complement and/or
12. Considerations in the development of pluripotent stem cellebased therapies424
III. Specialty biologics and indications
supplant the need to test in animals. Regulatory agencies have encouraged sponsors to attend
to the “3R’s” in nonclinical pharmacology/toxicology testing (replace, reduce, and refine)
and in vitro/in silico models may assist to that end (Wange et al., 2021).
Pharmacologic efficacy assessments
The purposes of animal pharmacologic efficacy/POC testing are to demonstrate the pos-
sibility of therapeutic benefit in patients, explore the doseeresponse relationship between the
cell product and desired outcomes, and potentially elucidate and provide support for the
mechanism of action. These studies are typically conducted in an animal model of the pro-
posed human disease or injury state and mimic the intended clinical route of administration.
The studies are also designed to gather information on the basic mechanisms of structure and
function, host/implant interface and systemic effects after implantation. Such observations
are particularly important for products that contain cells that are not terminally differentiated
at the time of implantation.
Study design, dose selection and justification of pharmacologic endpoints
Although pharmacology studies are not required to comply with GLPs, the burden to
demonstrate efficacy with PSCs preclinically should drive robust study designs that consider
the principles of GLP to support Phase 1 entry. These may include establishing standard
operating procedures (SOPs) for reproducibility of endpoints and procedures to reduce
observer bias (e.g., blinding observers who perform qualitative functional assessments
such as percent behavioral recovery in spinal cord injury). The design, number of animals,
and endpoints used in pivotal IND-enabling studies can be guided by information from
the literature along with data from pilot studies conducted in small cohorts of animals. There
is no rule or guideline governing appropriate level of power for pharmacology studies;
enough animals should be included in both placebo/sham control and treatment groups to
assure sufficient sensitivity and generate robust datasets should animals die on study. While
a single dose level, identified through pilot studies, is generally adequate to demonstrate
pharmacological activity or “proof of biology” in the model of choice, additional dose levels
will provide further information to aid in dose extrapolation for first-in-human clinical trials.
In particular, identification of the MED is helpful to determine a minimal threshold for antic-
ipated therapeutic effect. It is also common with cellular therapy that a maximally tolerated
dose (MTD) is replaced by a maximum feasible dose (MFD) as the tissue area or anatomical
compartment into which the cells are delivered may limit the full dosing range; indeed an
MTD may never even be identified due to physical constraints and lack of toxicities. Identi-
fying each of these target doses will help guide the proposed clinical dosing range. The dura-
tion of the study should be sufficient to establish the durability of the effect but also to gather
information about transplant/host integration and potential local and systemic effects that
might need to be considered when designing pivotal toxicity studies.
Study designs should strive to employ quantitative rather than qualitative endpoints as
they can reduce observer bias and allow comparability across experiments. These may
include biomarkers that quantify activity either directly (e.g., blood glucose) or indirectly
(e.g., C-peptide for active insulin processing, or mo tor performance by grip strength). Where
Nonclinical development 425
III. Specialty biologics and indications
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