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possible, the clinical measures that would be used in humans should be leveraged in animal
studies, as these will facilitate translation to the clinic.
Pharmacologic mechanism of action
In some cases, defining a precise mechanism of action for the cell therapy may be difficult
and may never be fully known. Understanding the mechanism of action, based on in vitro
and/or in vivo data however helps to optimize int erpretation of study data and facilitate clin-
ical development. For example, therapies that aim to replace a secreted product such as insu-
lin for diabetes or dopamine to treat Parkinson’s disease, can cite a clear ligand-receptor
mediated activity. In these instances, investigators may reference a body of scientific litera-
ture to elaborate a proposed mechanism of action, including demonstration that the animal
model has a homologous human receptor target and activity is mediated by a well-
characterized biological process.
Pharmacodynamics and pharmacokinetics
Pharmacodynamics
Pharmacodynamics examines the activity of a drug or therapy on the body. In the case of
cell therapies, the goals of these studies are to define a therapeutic index (effective dose), un-
derstand the duration of action, and explore undesirable effects (exaggerated pharmacology).
In many instances, carefully designed efficacy studies can include additional endpoints or
dose levels to support an understanding of the pharmacodynamic properties of implanted
cells.
Defining therapeutic index (effective dose) and exaggerated pharmacology
For biologics, the therapeutic index (TI) defines a range of doses (or dose multiples) that
produce desired efficacy up to the level at which undesirable or exa ggerated pharmacology
is observed. Examples of exaggerated pharmacology might include excess insulin production
from pancreatic islet cells that might trigger hypoglycemia, dyskinesias from cell integration
in Parkinson’s disease, or arrhythmias from ventricular cell wall implants.
Pharmacokinetics (absorption, distribution, metabolism, and excretion)
Pharmacokinetic (PK) absorption, distribution, metabolism, and excretion (ADME) are
concepts from drug development that have been adapted to cell therapy. In simple terms
these studies assess how the body responds to a therapy. PK-ADME studies are conducted:
(1) to establish correlations between dose/exposure and effects (i.e., desired activity), (2) to
provide data to ensure sufficient product exposure for toxicity studies, and (3) to provide
rationale for extrapolation to a safe human dose supporting a dose escalation scheme. For
PSC-based therapies these parameters are assessed by measuring the engraftment, bio-
distribution, cell trafficking, and persistence of the cells in vivo.
Engraftment and biodistribution studies are unique to cellular therapies and replace tradi-
tional toxicokinetic exposure studies conducted for new chemical entities (NCEs) and other
biotechnology products. The goals of these evaluations are to determine if the doses admin-
istered result in appropriate cell survival and exposure in the host for the effects measured in
12. Considerations in the development of pluripotent stem cellebased therapies426
III. Specialty biologics and indications
the toxicity study. Additionally, an assessment of distribution should determine the range
over which transplanted cells could migrate including trafficking to potential nontarget tis-
sues. Typically, an evaluation of both engraftment and biodistribution can be incorporated
into the general toxicity study. In these instances, assessments are primarily made as part
of the histopathological evaluation of the transplant site and complete organ panels in com-
bination with more sensitive molecular techniques such as PCR to detect human-specific gene
sequences.
Engraftment
Both the site of implantation and level of immunodeficiency of the animal model will
determine engraftment success. While most preclinical studies are likely to be conducted in
immunocompromised mice or rats, these model s are imperfect and the levels of site-
specific macrophages, dendritic cells (DC), and NK cells can still impact engraftment. Sites
inside the CNS carry some degree of immune privilege, whereas those outside are subject
to immune surveillance and graft rejection. For example, the subcutaneous space, which con-
tains large numbers of specialized antigen recognizing DC populations (Langerhans cells) has
a lower incidence of engraftment success that may limit biodistribution (Cooke et al., 2006;
Gutierrez-Aranda et al., 2010). By comparison, sites such as the heart, kidney, and liver,
which have low levels of DCs and respond relatively poorly to foreign antigens, are more
favorable for engraftment and PSC-based therapies engraft at lower doses (Cooke et al.,
2006). However, administration to these organs is likely to result in widespread bio-
distribution, which is generally undesirable. An understanding of local site-specific biology
can be used to justify dose-related engraftment success and explain any observed xenogeneic
rejection.
Assessment of engraftment in the toxicity studies can be leveraged to rationalize local
tolerance of the product. Importantly, the total dose of cells administered may dictate the
ability of a site to tolerate a transplant. Because of the small size of rodents, the maximal
dose that can be administered may be less than that intended for the clinic; however with
allometric scaling considerations, a reasonable assessment of local tolerance may still be
possible. Nevertheless, it may still be prudent to perform a separate acute local tolerance
study in an immune-suppressed large animal model. Information about implant/host inte-
gration and durability of engraftment can also drive decisions for re-administration of the
cell product. If this is a likelihood, investigators will need to justify tolerability for re-
administration.
Biodistribution (cell trafficking and persistence)
Because cells are a living therapy, and therefore never static, the dynami cs of cell prolifer-
ation, growth, migration, alterations in cell phenotype, and the kinetics of cell death need to
be characterized. These parameters are assessed either in dedicated biodistribution studies or
as part of the toxicity study. The data from these studies, together with an understanding of
the dose response, are key components when developing the rationale for the starting human
dose for a first-in-human trial.
Cell dose, study duration, and cell-labeling methods need careful consideration when
designing biodistribution studies. Multiple time points should be assessed to understand
how cells respond to the host environment over both the short and long-term. The new
Nonclinical development 427
III. Specialty biologics and indications
ICH guideline S12 on nonclinical biodistribution considerations for gene therapy products
(ICH, 2023), while directed to investigators developing gene therapeutics, is applicable to
ex vivo genetically modified cells, and has some useful considerations for cell therapy devel-
opers including (a) general considerations in the design of biodistribution studies,
(b) immune status of animal models, (c) assay methodologies, and (d) assessment of gonadal
tissues.
Various methods can be used for assessing cell distribution, including cell labeling and tis-
sue visualization in animals, together with ultra-sensitive PCR and ddPCR techniques. Ideal
labeling techniques will facilitate quantification of the target cells, have minimum transfer to
nontarget cells, be compatible with histochemical methods used for morphological analysis,
and not alter the biology of the cell product. The benefits and limitations of some current cell-
labeling techniques are discussed below.
Human-specific markers
Because donor PSC-derived cells are a xenotransplant in animals, human-specific markers
can be used to identify the transplanted cells. Human cells can be tracked in animal tissue by
qPCR for human-specific (e.g., Alu) sequences (Laflamme et al., 2007; Funakoshi et al., 2017).
These markers can be used alone or in combination with other cell-labeling techniques
including radioisotopes, fluorescent tracer dyes and markers of cellular proliferation, such
as 5-bromo-2-deoxyuridine (BrdU) and related molecules. While these techniques are highly
sensitive, they are terminal procedures and cannot be applied easily for in-life monitoring.
Moreover, tracers and markers of proliferation can be hampered by cytotoxic side-effects
of the labeling procedures, dilution, and loss of the marker over time as a result of cell divi-
sion. Thus, while useful for measuring the efficiency of acute delivery, these techniques may
offer minimal long-term information regarding cell survival, proliferation, and
differentiation.
Reporter genes, edited cells, and Y chromosomes
Viral transduction methods allow stable integration of reporter genes [e.g., green or yellow
fluorescent proteins (GFP; YFP), lacZ, alkaline phosphatase, firefly luciferase, etc.], facilitating
the evaluation of viable cells by fluorescence microscopy and flow cytometry. Reporter gene
imaging can be used to track long-term cell survival, migration, division and differentiation
and provide information on location and numbers of cells (Pearl and Wu, 2008; Wilson et al.,
2009; Hong et al., 2010). If a reporter gene is used in toxicity studies, investigators will have to
demonstrate that the reporting agent and/or technology used to add it to the cells is benign
and does not itself impact development of tumors from the product. Reporter genes can be
suitable for application in independent biodistribution studies where safety endpoints are
not being derived.
If the PSCs are edited, their presence may be readily determined by assaying fo r the
unique edited region in the genome. Similarly, if cells overexpress a gene of interest, such
as a gene to confer hypo-immunity, the cells may be tracked by probing for the presence
of the overexpressed gene through ultrasensitive sequencing techniques. Alternatively, if a
male PSC-derived line is used in female recipients, cells can be histologically labeled for
the Y-chromosome via in situ hybridization. Disadvantages of using reporter genes and
PCR methods are the requirement of a biopsy or terminal procedure for the performance
12. Considerations in the development of pluripotent stem cellebased therapies428
III. Specialty biologics and indications
of the cell tracking in tissues, and limitations related to sampling a portion of each tissue for
analysis of the entire tissue.
PET, SPECT, MRI, and bioluminescence
Various noninvasive (nonterminal) imaging modalities including positron emission to-
mography (PET), single-photon emission tomography (SPECT), magnetic resonance imaging
(MRI), fluorescence and, bioluminescence imaging (Wilson et al., 2009; Hong et al., 2010; Nar-
sinh et al., 2009 ) provide methods to visualize grafted cells, evaluate targeting of transplants
to specific areas, and monitor regenerative changes after cell therapy. PET, SPECT, and MRI
have the advantage in that they are all clinically relevant methods that may also be used to
monitor cells in patients (Hong et al., 2010). Despite more than a decade since the first pub-
lication of this chapter, MRI remains the most sensitive and useful of the three imaging mo-
dalities and is therefore the focus of this section.
MRI can be used to track cells labeled with contrast materials. The two classes of contrast
material that are generally used are gadolinium chelates and iron oxide nanoparticles
(Cromer Berman et al., 2011; Sykova et al., 2011). Pretreatment of these particles with stan-
dard transfection agents facilitates cellular internalizat ion into endosomes. Visualization of
the particles can be used to confirm cell delivery and monitor short-term migration into sur-
rounding tissu es. Despite the apparent simplicity of this method, it is uncertain whether
physiochemical properties of these labels affect cell differentiation or possess genotoxic po-
tential (Fayol et al., 2013; Crabbe et al., 2010).
The shortfall of most imaging methods is that they are unable to distinguish between
viable and nonviable cells. Nuclear labeling techniques (e.g.,
18
F-fluorodeoxyglucose PET)
can theoretically be used in combination with tracking methods to evaluate the anatomy of
the transplant in relation to its viability (Wu et al., 2013). Ultimately, multiple analytical
methods, including histopathology as well as newer more sensitive approaches to track
live cells, may be needed to best assess and monitor cell fate.
Special considerations for species and model selection for assessment of toxicity
For pharmaceuticals (and some biopharmaceuticals), toxicity studies are conducted in two
species, a rodent (mouse or rat) and a nonrodent (typically rabbit, dog, pig, or nonhuman pri-
mate). Two species are used to help inform dose extrapolation to the clinic but also to identify
the most sensitive species in which to determine the no-observed-adverse-effect-level
(NOAEL). The two-species concept is often impractical when applied to cell therapies, pri-
marily due to the lack of immunocompromised large animal species and the challenges
with maintaining long-term immunosuppressive regimens. If carefully rationalized, investi-
gators may be able to justify the use of only a single species for toxicity testing. Additional
considerations may arise if a novel delivery device will be utilized in clinical trials, which
may require testing in a nonrodent species with a larger body size for its characterization.
A variety of immunocompromised models have been discussed above. Relevant animal
model selection is based upon several considerations including the biological and structural
goal of the study, the injury or disease under study, the animal’s anatomy, physiology and
size, animal availability, housing requirements, cost and technical feasibility, and historical
experience with the model. Regardless, data must be provided to support the rationale for
model selection that demonstrate the sensitivity of the proposed animal model and its
Nonclinical development 429
III. Specialty biologics and indications
appropriateness for assessing safety. Investigators should ensure that the nonclinical section
of their regulatory submission carefully addresses the rationale for their selected animal
model for safety testing.
Disease/injury animal model versus healthy naive animal
For most drugs and biopharmaceuticals, Phase 1 testing in healthy human subjects impels
the conduct of typical toxicity studies in healthy naïve animals. Investigators conducting
pivotal toxicity studies for PSC-derived therapies will need to provide a rationale for using an-
imals that are either naïve or model the human disease. In most cases animal models of injury/
disease may be the most relevant model in which to assess not only POC but also safety. In an
effort to minimize animal use, hybrid pharmacology/safety studies may be considered when
technically feasible. Ofnote, vitality and life span of injury/disease models may be a critical lim-
itation to their use in hybrid studies given that GLP safety/toxicity/tumorigenicity evaluation
of cell therap ies are lengthy, most commonly at least 6 months in duration.
If disease models are used, the strengths and limitations of each animal model, including
the extent to which the model has been characterized, should be considered for extrapolating
findings to humans. For example, there can be differences in the types of tissue damage and
endogenous recovery mechanisms that occur following spinal cord injury in mice, rats,
nonhuman primates, and humans, and the implications of each model can affect translation
of the data to humans. Data will be needed to show that the model can be accurately and
reproducibly generated and that the cells can be delivered consistently to the target area
across individuals. In this context, it is important to understand the optimal window for de-
livery of cells postinjury. If the clinical indication is chronic, then chronic rather than acute
animal models should be used; however, in some cases these may not be available or techni-
cally feasible. In these situations, it is important to discuss the choice of model with the reg-
ulatory agency prior to conducting pivotal studies. A sufficient number of animals should be
included to assure statistically meaningful analyses at the end of the study, while accounting
for potential animal loss due to infections or spontaneous tumors (both of which are common
in immunocompromised animals), attrition due to surgical procedures, and other sponta-
neous health causes. In some study designs this can mean starting with as many as
50%e100% more animals than are sought for analysis at study termination. The death of
an animal on pivotal IND-enabling studies, including pharmacology studies, is scrutinized
with additional focus by regulatory authorities because of the implied safety signal for pa-
tients. If an animal death occurs during a study, it is important to have a postmortem eval-
uation by a board-certified toxicologic pathologist to identify a cause of death and determine
whether the death was related to the test article or not.
Safety pharmacology
In general, safety pharmacology needs to be considered on a case-by-case basis depending
on the specific characteristics of the cell-based product. Cells, and/or the pharmacologically
active substances they secrete, may have effects on the core physiological syste ms (CNS, car-
diac, and respiratory) but also secondary systems (renal, gastrointestinal, autonomic, im-
mune, skeletal muscle, and endocrine systems). Specific guidelines for the conduct of
safety pharmacology studies are provided in ICH S7A and S7B (ICH, 2000a,b). The ICH
core battery tests include specific assessments of the cardiovascular system, respiratory
12. Considerations in the development of pluripotent stem cellebased therapies430
III. Specialty biologics and indications
system, and the CNS. Specific safety pharmacology assessments needed for a particular cell
type, when considered appropriate and technologically feasible, are generally made as part of
the toxicology assessments rather than as stand-alone studies (Cavagnaro, 2007). For
example, neurological batteries can be added to a study assessing safety of a neural cell prod-
uct to ensure that core central and peripheral nerve functions are not negatively affected. Or,
cardiovascular performance including blood pressure, ejection fractions, and cardiac rhythm
might be assessed for a product treating a cardiovascular cond ition.
Toxicity assessments
General toxicity
Although guidance documents have been issued by regulatory agencies to provide assis -
tance in the assessment of nonclinical safety of cell products, developers should recognize
that the ultimate study designs including appropriate animal models will generally be
product-specific(Cavagnaro, 2007). Ideally, toxicology studies designed to support product
safety will be fully GLP-compliant. However, evaluation of safety in animal models of disease
may be difficult to conduct with strict adherence to GLP regulations, due to novel routes of
administration including novel surgical procedures, limited animal numbers, and the unique-
ness of animalmodels of disease that have limited historical data. In such cases, the principles of
GLPs can still be followed, and where deviations occur, they should be documented, evaluated
for their impact on the expected clinical application, and discussed with the regulatory agen cy.
The US FDA Guidance for Industry: S6 Addendum to Preclinical Safety Evaluation of
Biotechnology-Derived Pharmaceuticals (FDA, 2012) outlines seven key areas of potential
safety concern for cell therapy products including; (1) tissue reactions at the site of adminis-
tration, (2) potential inflammatory response in target and/or nontarget tissues, (3) host im-
mune response to cells, (4) migration from the site of administration, (5) potential to
differentiate into an unintended/inappropriate cell type, (6) unregulated/dysregulated pro-
liferation of cells within host, and (7) potential tumorigenicity. Developers must address these
issues directly in their safety/toxicity package with data that demonstrate their product is
either free of these features or if present, define the characteristics, limitations and potential
risk for patients. General strategies for tackling these issues and how they migh t be incorpo-
rated in a toxicity study design are discussed.
When nonclinical studies are conducted “in-house” and/or are reported in peer-reviewed
journals and intended to support clinical trials, they should be well-controlled and designed
to answer specific toxicological questions. The data should also be available in sufficient
detail to allow an independent review of the studies, including tabulated individual animal
data. Ideally, study designs should include not only efficacy but also toxic ological endpoints
such as defined clinical observations and clinical laboratory parameters, as well as macro-
scopic and micro scopic histopathological evaluation of tissues by a board-certified veterinary
pathologist. These points are discussed in detail below.
Justification of dose
Usually at least two to three dose levels and an appropriate control group are assessed for
safety, including the effective dose (ED), a multiple of the ED, and a maximum feasible dose
Nonclinical development 431
III. Specialty biologics and indications
(MFD). Investigators are encouraged to review previous nonclinical and clinical experience
with similar products to aid in estimating the nonclinical doses as well as justification for
the starting clinical dose. Doses can be extrapolated based on a denominator of body weight,
body surface area, target organ volume or area, or absolute number of cells being replaced,
depending on the route of administration.
Justification of delivery method
Investigators will need to include extensive details regarding the cell delivery device and
delivery procedure, such as surgical needs to access the site of administration, administration
strategy relative to lesion, number of injections, injection rate, injection volume, and needle
indwelling time. Even simple differences between devices in animal versus clinical studies
will need to be justified e.g., if a different gauge needle is used in animals than that intended
for the clinic, bench testing will be needed to show comparability (of cell recovery and
viability) between the nonclinical and clinical delivery procedures. More complex or novel
devices may need a separate review by CDRH (see section above).
Justification of test system
Considerations for selection of relevant models and species are discussed above. In many
cases only one relevant animal model of disease may be available to assess activity and/or
safety; in other cases, more than one model may be available and sufficiently characterized
to allow for meaningful extrapolation. Both male and female animals need to be used unless
the intention is to treat a sex-specific disease in patients.
Considerations for study design and endpoints
Toxicity study designs generally include early and late scheduled time points for examina-
tion to assess both acute/transient and chronic effects of the treatment (e.g., 1, 6, and
9 months). In some cases, it may be important to understand reversibility if there are adverse
effects. Studies should include appropriate controls (e.g., groups with a surgical control,
vehicle control, positive- or negative-control cells or an empty encapsulation device). Ideally
these studies should be appropriately powered with sufficient numbers of animals of both
sexes, in each dose group per time point, to assure statistically meaningful analyses at the
end of the study. For later time points, more animals may be needed to account for unsched-
uled deaths. Toxicity assessments should include: viability, weekly body weight, food intake,
mass palpation, clinical observations, organ weights, clinical chemistry (blood), hematology,
full organ histopathology, and any specific safety pharmacology assessment s required for a
particular cell product, as described above.
General toxicity studies should include a comprehensive assessment of cell engraftment
and survival at the site of administration for each animal. Such an evaluation may not neces-
sarily be fully quantitative but should provide some sense of the level of engraftment and
overall cell trafficking profile in each animal. The number of engrafted cells present should
be taken into consideration when interpreting the safety/functional outcome with
morphology outcome.
For all unscheduled deaths, necropsy and comprehensive histopathological evaluation
need to be conducted, including an attempt to determine the cause of death. Postmortem as-
sessments performed by a board-certified veterinary toxicologic pathologist should include:
12. Considerations in the development of pluripotent stem cellebased therapies432
III. Specialty biologics and indications
(a) comprehensive gross pathology examination, (b) oversi ght of the harvesting and weigh-
ing of a complete panel of organs/tissues and organ weight data interpretation, and
(c) histopathology of the target organ and all major organs and gross lesions.
Repeated dose toxicity studies are only relevant if the clinical use includes multiple
dosing. For example, in the case of pancreatic islet cells, toxicity studies should mimic
the c linical scenario of re-implantation if re-administration is planned for the clinic. In these
cases, both acute and cumulative effects should be considered as they might affect subse-
quent implants such as immune sensitizatio n to the product or scarring at the site of
administration.
The emergence of genetically modified cell products has heightened awareness of the need
to understand the long-term safety risks of these products. If investigators intend to use a
genetically modified cell product, they should engage early with the regulatory authority
to de-risk their plans for assessing genotoxicity and safety (see below for further discussion).
Reproductive and developmental toxicity
Traditional developmental and reproductive toxicity (DART) studies are generally not
applied to cell therapies. The need for any assessment of reproductive risk is dependent
upon the nature of the product, the clinical indication, and the intended patient population.
Consideration also depends upon any expressed products and/or observed biodistribution
e.g., if using ex-vivo modified cells that might secrete or block a protein of reproductive
risk such as an anti-VEGF. Effects to the reproductive system identified in biodistribution
and general toxicity assessments, which suggest a cause for concern must be addressed for
regulators. If studies are necessary, study designs might utilize a different dose and/or route
of administration in order to investigate specific concerns that have emerged in the general
toxicity/biodistribution studies. The animal model, dose level(s), and dosing frequency
should be selected to best test the hypotheses and understand potential clinical risks. It is
incumbent upon the sponsor to characterize risk for distribution to gonadal tissues or poten-
tial for germline transf er. This can begin with evaluating for presence of material in the go-
nads to ascertain if further testing is even warranted.
Genetic toxicity
As with protein-based biopharmaceuticals, the standard battery of genotoxicity studies is
generally not considered to be relevant for cell-based therapies, unless there is a specific cause
for concern. The advent of gene editing has now opened the door for the development of
genetically modified PSC products that may include specific edits to correct mutations or
‘cloak’ the cells from the immune system, i.e., hypo-immune cells. Gene editing can occur
with use of either (1) nuclease dependent ZFN’s, TALENS, CRISPR, or (2) nuclease indepen-
dent tools (base editing and synthetic triplex-forming peptide nucleic acids). These new prod-
ucts now require an assessment of genotoxic risk as the reagents used are known to interact
directly with DNA or other chromosomal material with the potential for both on- and off-
target editing to give rise to a carcinogenic cell. These new gene edited cell products are regu-
lated under a whole host of new guidances that offer a framework for nonclinical testing
including the new FDA guidance “Human Gene Therapy Products Incorporating Human
Genome Editing” (FDA, 2024b).
Nonclinical development 433
III. Specialty biologics and indications
Research has highlighted our lack of understanding of genomic outcomes associated with
double stranded (ds)-breaks from nuclease-dependent on-target editing including insertions,
deletions, and rearrangements that may cover megabases (Boutin et al., 2022; Lackner et al.,
2023). Covalent changes to DNA elicited by nuclease independent tools such as base editing,
may also generate intermediates that lead to ds-breaks (Fiumara et al., 2023). The conduct of
studies to specifically assess the genotoxic potential of the product fall into three broad
buckets: (1) safety risk assessment of manufacturing with process-related editing reagents
including: guide RNAs, enzymes, vectors, cell permeabilizing reagents etc.; (2) genotoxicity
assessment of both on- and off-target editing, as well as potenti al translocations, indel forma-
tion, genomic rearrangement s, and chromosomal stability; and (3) carcinogenic potential for in-
dependent cellular proliferation in the absence of normal growth regulatory signals. Multiple
methods and algorithmic approaches exist for assessing both on- and off-target editing with
no one method considered superior; most investigators tackle the challenge of assessing gen-
otoxic risk by using a multipronged approach of in silico and in vitro assessments. And, while
improvements in the fidelity of the editing reagents (e.g., new CAS enzymes, etc.) and
methods of editing hold promise for decreasing patient risk, a comprehensive genotoxicity
plan is still needed for any cell therapy that has undergone genetic editing. Early interaction
with the regulatory agency is advised to align on the genotoxicity plan.
Tumorigenicity assessment
One of the greatest concerns in the application of PSC-based therapies is their potential to
form tumors, particularly teratomas, which are tumors comprised of tissue from more than
one embryonic germ layer. Almost paradoxically, while these tumors are undesirable clini-
cally, the capacity to form a teratoma in vivo is a definitive criterion for establishing a cell
line as pluripotent (Gropp et al., 2012). While contaminating or residual PSC in the cell prod-
uct are of primary concern, tumorigenesis can be driven by many other factors including:
chromosomal instability, culture adaptation, the presence of hyperplastic progenitor cell pop-
ulations, host microenvironment and immune status (Bailey, 2012; Au et al., 2012). Demon-
strating that a PSC-derived product carries little-to-no risk for tumorigenicity should be
drawn from multiple lines of evidence from both in vitro and in vivo studies to provide a
“weight of evidence” (WOE) to argue that these tumors are unlikely to develop. In designing
these assays, sponsors will need to address several specific regulatory recommendations
described in the FDA “Guidance for Industry: Preclinical Assessment of Investigational
Cellular and Gene Therapy Products” (FDA, 2013), including: (1) Demonstrating stable
growth kinetic profile of the starting pluripotent population, (2) assuring chromosomal
and genomic stability of the starting population, (3) characterizing the differentiation status
and cellular attributes of the product, (4) establishing the threshold for residual undifferenti-
ated stem cells, (5) assaying the potential for tumor formation of the product, and (6) defining
the limitations of the assays for extrapolating risk in the target population. The section above
on Pluripotency and the Potential for Tumorigenicity provides a discussion of tumorigenicity
assays performed as part of product characterization.
Assaying potential for tumor formation in vivo
The potential for the injected cell product to proliferate into a tumor mass should be
assessed. Tumorigenicity studies may be conducted as standalone studies or assessments
12. Considerations in the development of pluripotent stem cellebased therapies434
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may be incorporated into a toxicity study. In general, tumorigenicity is assessed by injecting
the cell product into genetically immune-deficient or chemically immunosuppressed rodents
(mice or rats), with at least 10 animals per sex, per dose group, available for analysis at each
time point. The cell product candidat e is administered via the intended clinical route of
administration, and evaluation should include a long-term time point of at least 6 months
duration. A board-certified veterinary toxicologic pathologist should review tissue sections
to dete rmine whether teratoma formation has occurred. Pilot studies may be used to estimate
the tumor threshold and rate, providing a basis for designing the final study. Larger numbers
of animals may be enrolled at the start of a study to account for unscheduled deaths and
spontaneous tumors, particularly in immune-compromised rodents (Cavagnaro, 2007).
Ideally, a tumorigenicity study should use cells that are at the latest limit of cell culturing
to evaluate risks at the boundary of the manufacturing process. Usually, one or two dose
groups of the clinical-grade product are sufficient, including the use of an MFD. A short-
term pilot study is often performed to confirm initial cell engraftment; however, this study
will not ensure long-term engraftment at later time points. Investigators should ensure that
appropriate positive control group(s) are included in the pivotal study design and that the
study is appropriately powered to detect tumors, which are expected to be rare events. Con-
trol groups may include a cohort of animals that are administered the pluripotent starting cell
population, or groups into which known amounts of PSC are spiked at various dilutions into
the differentiated product. Whatever controls are chosen, investigators will need to justify the
choice. The final design should be vetted with the appropriate regulatory authority prior to
study initiation to ensure a costly study does not have to be repeated.
While undifferentiated stem cells are often considered to be the primary risk for teratoma
formation, there is the potential that other cell types, particularly progenitors of embryonal
tumors, could be present. These cell types could go unrecognized unless a targeted assess-
ment is conducted to ensure their absence. The engrafted cell product should be evaluated
for hallmarks of malignant progression including features associated with malignancy of hu-
man teratomas as these most closely parallel the tumors formed by PSCs. The histopathology
assessment should include review for the presence of primitive embryonic type cells, partic-
ularly neuroectodermal cells and extraembryonic tissues, and vascular space invasion con-
taining clumps of neoplastic cells (Cunningham et al., 2012). A board-certified pathologist
with specific training in the malignancy of teratomas can be engaged to provide an indepen-
dent expert opinion to support the toxicologic pathologist.
Local tolerability
Assessment of local tolerance and tissue compatibility of the clinical formulation is usually
evaluated as part of the animal model of efficacy and/or general toxicity study, thus obvi-
ating the need for separate local tolerance studies. The goal of this assessment is to improve
clinical efficacy and safety by exploring and optimizing both the method and site of cell de-
livery (implantation site). Many novel routes of administration have been employed depend-
ing upon specific product attributes and intended clinical indication, including: intracerebral,
intraspinal, intravitreal, intraocular, intramuscular, intraportal, intrarenal, intrasplenic, intra-
peritoneal, and subcutaneous. Each unique delivery route may pose an increased potential
for toxicity to tissues at or near its administration site, and care should be taken to include
review of surrounding tissues as well (e.g., draining lymph nodes).
Nonclinical development 435
III. Specialty biologics and indications
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