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\begin{document}

\title{Mining scientific advice reports on cell-based products; insight in the
non-clinical development program.}



\author[1]{Tineke van den Hoorn}%
\author[2]{Tahira Nakchedi}%
\author[2]{Charlotte de Wolf}%
\author[2]{Marjon Pasmooij}%
\author[1]{Jan Willem van der Laan}%
\author[1]{Carla Herberts}%
\affil[1]{Medicines Evaluation Board}%
\affil[2]{Affiliation not available}%


\vspace{-1em}



  
  \date{June 18, 2024}


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\selectlanguage{english}
\begin{abstract}
Aims The field of cell-based therapies for human diseases is currently
evolving from promising treatment options to established therapeutic
concepts. The design of the non-clinical development program for
cell-based products, intended to provide a rationale for treatment and
to gain insight into the safety profile, is challenging because of
limitations caused by species-specificity. The elements of the
non-clinical package for cell-based products were evaluated using advice
reports from the European Medicines Agency database from 2013-2018 to
identify the approach followed for non-clinical development of these
products. Methods The purpose of the in vivo studies was designated to
be (a combination of) pharmacology/proof-of-concept, safety,
biodistribution and/or tumourigenicity. For biodistribution and
tumourigenicity also the need for, type and design of in vitro and in
vivo studies were recorded. Results In vivo studies for cell-based
therapies were primarily aimed at proof-of-concept (75/86), followed by
addressing safety (64/86), biodistribution (49/86) or tumourigenicity
(46/86). No animal studies were performed or proposed by sponsors or
regulators for six (out of 86) products, which contained cell types that
have been studied in humans for a relatively long time. For one-third of
the products in vivo biodistribution and/or tumourigenicity studies were
not considered necessary. In vivo tumourigenicity studies were regarded
of limited value. Conclusions Compared to more conventional medicinal
products, the non-clinical development program for cell-based products
was more tailored and focussing on proof-of-concept. For tumourigenicity
an in vitro approach may suffice. Total omission of in vivo studies
appears to be possible for products with sufficient clinical experience.%
\end{abstract}%



\sloppy


\textbf{}

\textbf{Introduction}

Cell-based therapies are at the forefront of drug innovation. They are
highly science-driven, involve innovative drug manufacturing processes
and offer potential curative treatment options for diseases, which
currently have no or limited treatment options {[}1{]}. Hematopoietic
stem cell transplantations, as well as adoptive T-cell-therapies and
dendritic cell-based therapies, have been used for decades. However,
only recently cell-based therapies have evolved from promising treatment
options to products for which the therapeutic effect has been
established {[}2,3,4,5{]}. Since the introduction of the advanced
therapy regulation in the EU in 2007 {[}6{]} cell-based therapies are
regulated as advanced therapy medicinal products (ATMPs) Given the
innovative nature of cell-based products and their high variability,
development often involves a case-by-case approach {[}7{]}. This
particularly relates to the non-clinical development program, which is
expected to provide a rationale for entering clinical development of a
medicinal product and to gain insight into its safety profile. To this
end, \emph{in vitro} and \emph{in vivo} animal studies are performed, in
which the characteristics of the product and the targeted disease
determine the developmental approach to a large extent.

Designing a non-clinical development program for human cell-based
products is complex, because species-specificity limits the value
of\emph{in vivo} animal studies. Consequently, \emph{in vivo} animal
studies with the intended human cell-based product (heterologous model)
may not always be relevant. While an animal study with the animal
equivalent cells (homologous model) may be informative, some insights
into the safety and efficacy of a cell-based product can only be
obtained when the product is tested clinically {[}8,9,10{]}.

While some guidance on the non-clinical development program for
cell-based therapies is available in regulatory guidelines {[}11,12{]}
these are not very prescriptive, as flexibility is needed given the
large variability in the types of products. For example, these
guidelines state that conventional non-clinical pharmacokinetic studies
are not relevant, while animal studies on cell migration
(biodistribution) and persistence of the therapeutic cells are generally
expected. Presumably, because these data are considered to be of value
for the characterisation of the risk profile of the cells and the
understanding of their potential therapeutic effect. Still, following
the fate of cells \emph{in vivo} is challenging. There are many
techniques that can be used to detect specific cells in the body, from
basic tissue sampling and subsequent detection of cells by
immunohistochemistry (IHC) or specific deoxy nucleic acid (DNA)
fragments by polymerase chain reaction (PCR) {[}13{]} to highly
sophisticated techniques of \emph{in vivo} cell tracking {[}14{]}.
Nevertheless, they all have their own pro's and con's, and no guidance
is provided on which methods are considered most informative.

Regarding the oncogenic potential of cell-based products, the guidelines
state that conventional carcinogenicity studies, in which the ability of
a medicinal product to transform host cells into tumourigenic cells is
evaluated in non-clinical models {[}15{]} are not considered appropriate
for cell-based therapies {[}11{]}. Instead, the potential for
tumourigenicity, i.e. tumour formation of the administered (grafted)
cells {[}16{]} should be considered and addressed as appropriate.
Several approaches to study the tumourigenic potential of a cell-based
product can be envisioned. These include an \emph{in vivo} assessment of
the human cell-based product in animals but also \emph{in
vitro}assessment of, for example, genetic stability (e.g. by karyotyping
and FISH {[}17{]}, the presence of transformed or undifferentiated cells
in the product (e.g. flow cytometry and qRT-PCR {[}18{]}, or the ability
for anchorage or growth factor-independent growth {[}19{]}.

To increase the insight in the chosen approach for the non-clinical
development for cell-based products we analysed the non-clinical data
package of cell-based medicinal products in scientific advice reports
from 2013 until 2018 provided by the European Medicines Agency (EMA). In
particular, the number and purpose of \emph{in vivo} animal studies was
analysed to gain better insight in the prevailing view of sponsors
and/or on the relevance of \emph{in vivo} animal data. Furthermore, as
biodistribution and the potential for tumourigenicity are most difficult
to study in humans, because of the complexity of detection of the
administered cell in a patient {[}20{]}) and the need for long-term
follow up for the assessment of tumourigenic risks {[}21{]} we
particularly focussed on the need for and type of studies addressing
these aspects in the non-clinical development program.

\textbf{Methods}

For the analysis of the non-clinical data packages for cell-based
products, we collected scientific advice reports on ATMPs, written
between January 2013 and July 2018, from the EMA database. We selected
products containing cells (including genetically modified cells) for
which the information on the non-clinical development program was
available in the advice reports and, if available, from the accompanying
briefing books. For all products, it was recorded whether dedicated or
combined studies addressing pharmacology, safety, biodistribution and/or
tumourigenicity had been performed. In case no studies were performed to
address biodistribution and tumourigenicity, it was recorded for what
reason: not necessary, planned or whether they were requested by the
regulator.

Regarding the biodistribution studies, it was scored per product whether
distribution to target and non-target tissues was assessed, whether
persistence of the cells was analysed and if quantitative techniques
were used. When available, it was noted whether the sponsor regarded the
study of value for proof-of-concept and/or safety.

Regarding the tumourigenicity studies, the number and type of studies
were recorded. For the \emph{in vitro} studies, the method was scored.
For the \emph{in vivo} studies the following aspects on the design of
the studies were noted as well: species, cell dose, administration route
and duration.

Analyses of the scored information were performed for all products and
for classes of cell-based products when categorisation was applied based
on the cell source (autologous versus allogenic) and the presence or
absence of genetic modification.

More detail on which data were acquired and how these were analysed is
provided in the Appendix.

\textbf{Results}

\emph{Non-clinical package}

In total 218 ATMP advice reports from 2013 until 2018 were collected
from the EMA database, containing information on 153 different products.
Of these, 59 were gene therapy medicinal products (GTMPs) not consisting
of cells, which were excluded from the study. One product was excluded
from the database, because it contained xenogeneic cells and seven
products were excluded, because information on the non-clinical program
was lacking. The remaining 86 products were categorized based on cell
origin and on genetic modification status (Figure 1). The majority of
cell therapies in development for which advice was requested during the
2013-2018 period, were not genetically modified (n= 60, 70\%). Of
these,31 were of autologous origin and 29 were of allogenic origin. The
majority of the genetically modified cells were autologous of origin
(n=20, 23\%) and only six (7\%) products were genetically modified,
allogenic cells.

In total 234 \emph{in vivo} studies were performed for 80 of the 86
products, and for some products additional animal studies were
envisaged. The six products for which \emph{in vivo} animal studies were
not conducted, planned or requested did not contain genetically modified
cells (data not shown). Pharmacology, which mainly accounts for
proof-of-concept (PoC), was studied for most of the products, followed
by safety (Figure 2). The number of products with biodistribution and
tumourigenicity animal studies was slightly lower. For these types of
studies information on biodistribution (8 products) and tumourigenicity
(14 products) was not always present in the non-clinical data package
provided by the sponsor. It is not known whether sponsors did not regard
studies addressing biodistribution and tumourigenicity of value for
their product or whether the product was not yet in the stage of
development that the issue of biodistribution and/or tumourigenicity
study would be relevant to address.

In guidelines for ATMPs it is recommended to combine the various
endpoints addressing e.g. pharmacology and safety in one study if
possible and sensible, resulting into a so-called combination study
{[}7{]}. Across the different types of \emph{in vivo} studies,
pharmacology was most often evaluated in dedicated studies, whereas
safety was often studied in combination studies (Figure 3).

\emph{Biodistribution}

For at least half of the products the sponsor regarded an \emph{in
vivo}animal study of value for the evaluation of the biodistribution of
their product, as for 49 of 86 products (57\%) a biodistribution study
was either conducted (n=40; 47\%) or planned (n=9; 10\%) (Table 1). For
25 products, the non-clinical data package contained two or more
\emph{in vivo} biodistribution studies. For the products (n=37; 43\%)
for which a biodistribution study was not conducted nor planned, this
was agreed upon by the regulators either immediately (n=21) or when
supported by further justification (n=3). For five products (6\%) the
regulators requested a biodistribution study in animals. For eight
products (9\%), a discussion on the biodistribution was lacking in the
sponsor's documentation and was not discussed by the regulators in the
advice report. Thus, for 24 of 86 products (28\%), both sponsors and
regulators considered there was no need for \emph{in vivo} studies on
biodistribution. There were no large differences in the presence or
absence of \emph{in vivo} biodistribution studies between the product
categories (Table 1). Even though for most products biodistribution was
studied in a dedicated study (30 of 49 products), sponsors indicated
that the results were of value for both understanding the PoC of their
product and its safety profile. For several products the value of the
biodistribution study for PoC-only or for safety-only was recognized
(respectively n=4 and n=11).

For nine products biodistribution was studied in healthy and for twelve
products biodistribution was studied in diseased animals. For nine of
these it was explicitly noted that the model was homologous. For three
products no information was present on the model and for the remaining
25 products immunocompromised animals, reflecting heterologous models,
were used. For 42 products information on the method to evaluate
biodistribution was provided. The most commonly used techniques were
IHC, quantitative-PCR (qPCR) or reverse-transcriptase PCR (RT-PCR),
Fluorescence \emph{In Situ} Hybridization (FISH), and microscopy. For
all these techniques tissue samples need to be harvested to allow for
detection of the cells. The more sophisticated techniques for studying
cell-distribution \emph{in vivo} (such as Positron emission tomography
(PET), Single photon emission computed tomography (SPECT), Magnetic
resonance imaging (MRI) and Bioluminescence imaging (BLI)) were only
used to study biodistribution for three products.

The vast majority of biodistribution studies provided information on the
presence of the administered cells in/migration to both target and
non-target tissues (40/49; 82\% of the products with biodistribution
studies, Figure 4). Only for four (autologous cell) products, sponsors
considered the option of assessing target tissue only (8\% of the
studies). For five products it was not mentioned which tissues were
assessed (10\%). Information on the relative distribution of the
administered cells was available for 42 (86\%) of the 49 studies,
suggesting that the method to determine biodistribution of the
therapeutic cells was also (semi-)quantitative.

Information on persistence of the administered cells was available for
44 products (51\%). For another 10 products (12\%) sponsors planned to
evaluate \emph{in vivo} persistence in the future, whereas for 13
products (15\%) sponsors did not aim to address it (Figure 5). For the
remaining 19 products there was nothing mentioned regarding persistence
in the submitted information.

\emph{Tumourigenicity}

Remarkably, while FDA guidance {[}11{]} specifically stipulates that for
tumourigenicity studies the intended clinical product should be used
(and not analogous animal cells), tumourigenicity was studied in a
homologous model for eight products of which three were studied in
healthy and five in diseased animals. For one product it was not
mentioned and for remaining 37 products immunocompromised animals,
reflecting heterologous models, were used. The chosen route of
administration (RoA) was most often intravenous or subcutaneous and
mainly a dose of 1x10\textsuperscript{6} cells was applied (Table 2).
For two products, various doses were used and for ten products the
administered dose in the study was not mentioned (most often for planned
studies). While there was some variability in the duration of
the\emph{in vivo} tumourigenicity studies, a study duration between
three and six months was noted for the majority of the products (n=28 of
46).

For most products the tumourigenic potential was addressed by
experimental data (\emph{in vitro} and/or \emph{in vivo} studies), only
for 10 products (11\%) sponsors did not investigate the tumourigenic
risk with any experimental data. Of note, for 14 (16\%) products there
was no information on tumourigenicity studies in the documentation. For
nine products (10\%), the risk for tumourigenicity was investigated
by\emph{in vivo} studies only, for 18 products (20\%) the tumourigenic
risk was only evaluated by \emph{in vitro} assays, and for 37 products
(43\%) both \emph{in vivo} and \emph{in vitro} studies were used to
address this potential risk (Figure 6). Thus for 46 of the 86 products
in the database tumourigenicity was studied and/or planned to be
studied\emph{in vivo} (Figure 2). For 18 of the 46 products with
\emph{in vivo}tumourigenicity studies, more than two \emph{in vivo}
studies tumourigenicity studies were part of the development package.
This often concerned the combination of a dose range finding study and a
pivotal study, or the assessment of tumourigenic risk upon various
routes of administration.

For 16 of the 40 products for which no \emph{in vivo} study was
performed or planned, it was explicitly agreed that an \emph{in
vivo}study was not necessary, while for six products regulators
indicated that the absence of \emph{in vivo} studies could be acceptable
provided that the company justified the absence of such a study. This
was apparently most often the case for the genetically modified products
(Table 3). For four products an \emph{in vivo} animal study was
explicitly requested. Products for which tumourigenicity was studied
with only \emph{in vitro} studies most often contained genetically
modified cells (Figure 6). For products containing allogeneic
genetically modified products, tumourigenicity was always discussed

The \emph{in vitro} tumourigenicity studies in the 55 (64\%) products
most often consisted of karyotyping (n=28) and/or colony
formation/proliferation assays (n=22). For 22 products, multiple types
of \emph{in vitro} studies were performed (Table 4).

\textbf{Discussion}

To gain insight into the non-clinical development program for cell-based
products we analysed the non-clinical data package of 86 different
cell-based products based on scientific advices provided by the EMA
between January 2013 and June 2018. A 5-year time period was chosen to
ensure that our data contained a sufficient number and diversity of
cell-based therapies to allow for a meaningful analysis of the
non-clinical development program. In comparison, a previous analysis of
the non-clinical program in EMA advices on ATMPs contained 54 ATMPs of
which thirteen were somatic cell therapy and eight were tissue
engineered products{[}22{]}.Within the 86 products analysed, a variety
of autologous or allogenic cell-based products with and without
genetically modifications were included, however the class of products
containing genetically modified allogenic cells was only represented by
six products.

A non-clinical development program consists of \emph{in vitro}
and\emph{in vivo} studies designed to provide a clinical treatment
rationale (PoC) and to gain insight into the safety profile of a
medicinal product. For cell-based therapies translation of animal data
towards the human situation has significant limitations. When testing a
human cell-based product in an animal (heterologous model) limitations
include possible differences in cell size or cell metabolic rate
{[}23{]}, immunogenicity (xeno-reactions can occur extremely rapid and
vigorous) {[}24{]} and potential species specificities in cell-cell or
cell-environment interactions {[}25{]}. These limitations all need to be
considered when interpreting study results. A homologous model in which
the animal equivalent of the human cell-based product is tested, could
be considered as an alternative \emph{in vivo} testing strategy.
However, interpretation of results from such a model may be complicated
by differences in manufacturing processes of the animal equivalent,
deviation of animal response from the human response or absence of an
appropriate animal equivalent of the human cells{[}25{]}.

Interestingly, while the translation of animal data to the human
situation has significant limitations for cell-based products, only for
six products animal studies were not performed or proposed by the
sponsors nor requested by the regulators. These products contained cell
types, which have been studied in humans for a relatively long time,
such as dendritic cells {[}26{]}, antigen-specific T-cells {[}27{]} and
mesenchymal stromal cells (MSC) {[}28{]}. Apparently, for these products
it was considered that additional animal studies would not provide new
insights on these types of products. As the clinical experience with
cell-based products is increasing, the knowledge on the (potential)
effects of the studied cell types is also increasing. This may result
into a reduction or even omission for the need of \emph{in vivo} animal
studies for certain types of cell-based therapies.

Still for most products the sponsors considered data from animal studies
of value for the development of their product. This was particularly the
case for studies on PoC as \emph{in vivo} pharmacology studies were
performed for almost all the products. Also safety was studied \emph{in
vivo} in animals for the majority of the cell-based products, albeit
more often in combination with other endpoints e.g. by also addressing
the cellular biodistribution or by including the safety endpoints in
pharmacology studies. Thus, it seems that the focus of the non-clinical
development program for cell-based products is more on pharmacology/PoC
than on safety. This is different from what we see for more
`conventional' medicinal products, where safety is an important
component of the non-clinical \emph{in vivo} studies and almost
exclusively evaluated in dedicated toxicity studies.

The fact that pharmacology is most often studied in a dedicated study
could be explained by a difference in timing of the studies, as
non-clinical data are mostly intended to support the rationale for
further development before safety studies are performed to ensure the
safety of the first clinical trial subjects. Pharmacology studies do not
require GLP compliance, in contrast to safety studies. This may have
contributed to the higher number of products with pharmacology studies.
Notably, although GLP-compliance is formally required for non-clinical
safety studies for human medicinal products, for cell-based therapies it
has been acknowledged that GLP compliance of \emph{in vivo} safety
studies may not always be feasible {[}29{]}.

In our analysis we particularly focussed on the need for and the type of
biodistribution and/or tumourigenicity studies since these aspects are
very difficult to study in humans. For more conventional medicinal
products, the kinetics of the product and its safety profile is studied
in animals. Interestingly, in our analysis \emph{in vivo} animal studies
on biodistribution and/or tumourigenicity were not considered necessary
by both sponsors and regulators for approximately one third of the
cell-based products irrespective of product type. This observation
suggests that for cell-based products a more tailored non-clinical
development program is necessary. Thereby sponsors should focus on the
need to better characterise the properties of the cell-based product and
consider the (im)possibilities of studying a products behaviour in an
animal model.

Even though a considerable number of cell-based product in our database
are lacking an \emph{in vivo} study on biodistribution, still this type
of study was performed for two-thirds of the products and considered to
be of value for understanding the PoC and to gain insight into the
safety profile. For biodistribution most often a heterologous animal
model was used. This is remarkable as cell migration is expected to be
dependent on chemotactic signals and cell-cell interactions which might
be species-specific and may thus better be studied in a homologous
model. Next to distribution to target and non-target tissue, also
persistence of the cells was (proposed to be) evaluated for
approximately two third of the products, suggesting that sponsors
already acknowledge persistence as an important biodistribution endpoint
to be investigated. Detection of administered cells in target and
non-target tissues was most commonly done by tissue sampling followed by
(semi)quantitative detection of these cells with various sensitive
techniques. Notably, more sophisticated techniques based on PET, SPECT,
and MRI imaging are being developed, which can trace living
cells\emph{in vivo} {[}14,20{]}, allowing for serial sampling and
whole-body scanning. However, these techniques were rarely used to study
the biodistribution of the products in our database. Possibly these
techniques were still not sufficiently developed at the time of the
design of the non-clinical studies for our analysed products. It is also
possible that these techniques were not (yet) attractive due to high
costs, limited access and/or the perception that these techniques are
too novel and have not yet proven their value to ensure their
acceptability by regulators. Notably, these newer techniques of cell
tracking may also become suitable for the evaluation of biodistribution
of cellular therapeutics in humans, thus in the clinical setting.
Clinical biodistribution studies would be of much more relevance as the
therapeutic product can be tested directly in the target `species'. The
hope is that these more advanced methods analysing distribution of
cellular therapies, will be implemented in due time and that they could
possibly obviate the need for extensive evaluation of \emph{in
vivo}biodistribution of human cell-based products in animals.

As tumourigenicity is a concern associated with cell-based products, it
is not surprising that for most products experimental data was used to
address the potential for tumourigenicity. For half of the products this
was planned or performed to be addressed by means of \emph{in
vivo}animal studies. For four-fifths of these products, \emph{in
vitro}studies supplemented the tumourigenicity evaluation. One-fifth
thus leaned on \emph{in vivo} studies only. The design of these \emph{in
vivo} studies appeared rather similar across the various products, and
all tended to follow the WHO guidance {[}16{]} on the assessment of
tumourigenicity of mammalian cells (i.e. 1x10\textsuperscript{6} cells,
subcutaneous administration, 3 to6 months in duration), except that
intravenous administration was often used as well to better reflect the
clinical RoA. Interestingly, this WHO guidance was developed for
characterisation of a master or a working cell bank (MCB, WCB) {[}16{]},
and not to address the tumourigenic potential of human cell-based
products. The suitability of this study design for evaluation of the
tumorigenic risk of cell-based products has not been established.
Recently, the added value of \emph{in vivo} tumourigenicity studies has
been questioned {[}21{]}. With respect to this, it is interesting that
only for a minority of products the \emph{in vivo} study was considered
to have some relevance by the regulators or experts. Despite the limited
relevance, the non-clinical data package on the tumourigenic potential
was considered sufficiently informative for most of the products. This
implies that while limited value was given to the \emph{in vivo}studies,
the value of \emph{in vitro} studies for the assessment of
tumourigenicity is recognized. Notably, an \emph{in vitro} -only
evaluation of tumourigenic risk was accepted for two types of products,
i.e. for autologous cells and for genetically modified products. For the
genetically modified products \emph{in vitro} evaluation of insertion
site analyses (ISA) seem to be sufficient for addressing the
tumourigenic risk. Possibly, the need for \emph{in vivo} animal studies
for the safety assessment of cell-based products may be further reduced
and more sensitive and standardised \emph{in vitro} assays may be
developed to characterise the risk for tumourigenicity {[}18,21,30{]}.

To conclude, our analysis has given insight into the non-clinical
development program for cell-based products, specifically on the number
and types of studies performed for the various types of products. It
appears not possible to define a common route to-be-followed for the
non-clinical development program of cell-based therapies. While it is
clear that during development studies on the pharmacology,
biodistribution, general safety and tumourigenicity should be
considered, it is not evident that for all these aspects animal studies
need to be performed or how they should be designed. Not only because
the variety of products prohibits a-one-size--fits-all approach for
non-clinical development, but also because insights are changing and
clinical experience is growing, which can affect the need for animal
studies for future products. Moreover, technical possibilities to study
certain aspects may change in time as well. Therefore, the
recommendation remains to tailor the non-clinical development program
for cell-based products to the specific need for information, depending
on the characteristics of the product and take into consideration
available knowledge and relevance of \emph{in vivo} animal studies. To
this end, early dialogue between sponsors and regulators will remain
utterly valuable.

\emph{Acknowledgement section}

\emph{Not applicable}

\emph{Conflict of interest statement}

\emph{Not applicable}

\emph{Funding information}

\emph{Not applicable}

\emph{Data availability statement}

\emph{Authors cannot share data for reasons of confidentiality, which
may fall under `embargo on data due to commercial restrictions' and
`data subject to third party restrictions.}

\emph{References}

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\end{enumerate}

\emph{Tables}

Table 1: Availability of biodistribution studies in the non-clinical
development\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& Performed & Planned & Requested & Absence to be justified & Not
necessary & Not mentioned\tabularnewline
\midrule
\endhead
Autologous not genetically modified (n=31) & 11 & 3 & 3 & 0 & 10 &
4\tabularnewline
Allogeneic not genetically modified (n=29) & 17 & 3 & 0 & 2 & 6 &
1\tabularnewline
Autologous genetically modified (n=20) & 9 & 3 & 1 & 0 & 4 &
3\tabularnewline
Allogeneic genetically modified (n=6) & 3 & 0 & 1 & 1 & 1 &
0\tabularnewline
\textbf{Total of biodistribution studies} & \textbf{40} & \textbf{9} &
\textbf{5} & \textbf{3} & \textbf{21} & \textbf{8}\tabularnewline
\bottomrule
\end{longtable}

Table 2: Design of the \emph{in vivo} tumourigenicity studies, regarding
applied dose, route of administration and duration\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllll@{}}
\toprule
& Dose & Dose & Dose & Dose & Dose & Route of administration & Route of
administration & Route of administration & Route of administration &
Duration (months) & Duration (months) & Duration (months) & Duration
(months)\tabularnewline
\midrule
\endhead
& \textbf{10e5} & \textbf{10e6} & \textbf{10e7} & \textbf{various} &
\textbf{n.m.} & \textbf{S.C.} & \textbf{I.V.} & \textbf{Other} &
\textbf{n.m.} & \textbf{0-3} & \textbf{3-6} & \textbf{6+} &
\textbf{n.m.}\tabularnewline
Autologous not genetically modified (n=17) & 3 & 6 & 2 & 0 & 6 & 5 & 3 &
4 & 4 & 5 & 9 & 1 & 3\tabularnewline
Allogeneic not genetically modified (n=19) & 1 & 11 & 5 & 1 & 2 & 11 & 6
& 6 & 3 & 3 & 11 & 5 & 3\tabularnewline
Autologous genetically modified (n=12) & 5 & 5 & 0 & 0 & 2 & 1 & 4 & 4 &
0 & 2 & 5 & 5 & 0\tabularnewline
Allogeneic genetically modified (n=3) & 1 & 0 & 1 & 1 & 0 & 1 & 0 & 2 &
0 & 0 & 3 & 2 & 0\tabularnewline
\textbf{Total46} & 10 & 22 & 8 & 2 & 10 & 18 & 13 & 16 & 7 & 10 & 28 &
13 & 6\tabularnewline
\bottomrule
\end{longtable}

\emph{n.m. = not mentioned, I.V. = intravenously, S.C.= subcutaneously}

Table 3: Availability of \emph{In vivo} studies for tumourigenicity in
the non-clinical development plan\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& Performed & Planned & Requested & Absence to be justified & Not
necessary & Not mentioned\tabularnewline
\midrule
\endhead
Autologous not genetically modified (n=31) & 11 & 5 & 2 & 1 & 6 &
6\tabularnewline
Allogeneic not genetically modified (n=29) & 14 & 4 & 0 & 1 & 5 &
5\tabularnewline
Autologous genetically modified (n=20) & 8 & 1 & 1 & 3 & 4 &
3\tabularnewline
Allogeneic genetically modified (n=6) & 3 & 0 & 1 & 1 & 1 &
0\tabularnewline
\textbf{Total of tumourigenicity studies} & \textbf{36} & \textbf{10} &
\textbf{4} & \textbf{6} & \textbf{16} & \textbf{14}\tabularnewline
\bottomrule
\end{longtable}

Table 4: Overview of the types of\emph{in vitro} tumourigenicity studies
performed within the four product categories\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& Karyotyping & Colony formation & Genomic analysis & Immortali-zation &
Other & Multiple studies\tabularnewline
\midrule
\endhead
Autologous not genetically modified (n=13) & 9 (69\%) & 9 (69\%) & 4
(31\%) & 2 (15\%) & 2 (15\%) & 8 (62\%)\tabularnewline
Allogeneic not genetically modified (n=21) & 14 (67\%) & 10 (48\%) & 5
(24\%) & 4 (19\%) & 3(14\%) & 11 (52\%)\tabularnewline
Autologous genetically modified (n=16) & 2 (13\%) & 3 (19\%) & 2 (13\%)
& 10 (63\%) & 1 (6\%) & 2 (13\%)\tabularnewline
Allogeneic genetically modified (n=5) & 3 (60\%) & 0 (0\%) & 3 (60\%) &
0 (0\%) & 0 (11\%) & 1 (20\%)\tabularnewline
\textbf{Total (n=55)} & 28 (52\%) & 22 (40\%) & 14(25\%) & 16 (29\%) & 6
(11\%) & 22(40\%)\tabularnewline
\bottomrule
\end{longtable}

\emph{Of note; the number of specific in vitro analysis used per product
category was calculated as a percentage of the total products in that
category}

\par\null

\emph{Figure legends}

Figure 1: Overview of selection process of scientific advices. All
advice reports from the EMA from 2013 -2018 were collected and the
advice reports on products containing human cells for which non-clinical
data information was present in the reports were selected. ATMP:
advanced therapy medicinal products.

Figure 2: Type of studies performed for the various product classes. In
total 234 \emph{in vivo}studies were performed for 86 products. The
purpose of the studies is shown for the products that are categorized
based on cell origin and genetic modification status that is depicted by
the different colours.

Figure 3: Percentage of dedicated and combination studies. The number of
\emph{in vivo} animal studies is related to the total amount of products
(86) addressing pharmacology (75), safety (64), biodistribution (49) and
tumourigenicity (46).

Figure 4: Migration analysis in biodistribution studies. The migration
towards target tissue only, or both to target and non-target tissues was
recorded for the biodistribution studies and specified for product
categories based on their cell origin and genetically modification
status.

Figure 5: Analysis of the persistence of the cells in the
biodistribution studies. The persistence of the cells in the animal was
either evaluated (Yes), not evaluated (No) or planned (Planned) to be
evaluated. For some products persistence of cells was not mentioned in
the advice reports (Not mentioned).

Figure 6: Approach for estimation of the tumorigenic risk. The approach
for evaluation of the tumourigenic risk by \emph{in vivo} and/or
\emph{in vitro} studies or none of these (`\emph{In vivo', `In vitro'} ,
or both \emph{`in vivo \& in vitro',} or `none'). For some products, the
tumourigenic risk is not addressed in the scientific advice (`Not
mentioned').

\emph{Appendix}

\textbf{Method for processing and analysing of scientific advice
reports}

A scoring table and a partly overlapping analysis table were designed to
record and analyse the information on the non-clinical development
program as present in scientific advice reports and corresponding
provided documentation from the sponsor. Both tables consisted for three
parts 1) general data and information regarding \emph{in vivo} animal
studies, 2) biodistribution studies and 3) tumourigenicity studies. For
a coded descriptive the options are given in between brackets. For an
open descriptive this is listed as `specified' in between the brackets.

\emph{Scoring table}

\emph{Part 1: Elements scored regarding general and in vivo data}

Advice number; Product name; Indication; Product type; Cell (and vector)
type; First and second advice coordinator; Topic(s) of question(s);
Occurrence of discussion meeting; \emph{in vivo} studies (no, yes, not
mentioned); Goal of \emph{in vivo} studies (Proof of Concept (PoC),
Biodistribution, Tumourigenicity, Safety).

\emph{Part 2: Elements scored regarding biodistribution studies}

Availability \emph{in vivo} study (no, yes); Reason for absence of the
study (not necessary, planned, requested, not necessary but
justification required, not mentioned); Analysis (clinical,
non-clinical) Species used (specified); Animal model used (healthy,
disease model, immune-compromised); Dose used (specified); Method for
labelling and detection (specified); Sampling time points (specified);
Site of Administration (specified); Migration (target tissues, both
target and non-target tissues); Quantifiable method (no, yes, not
mentioned); Persistence studied (no, yes, planned, not mentioned);
Informative (no, yes); Informative for (PoC/Mechanism of Action (MoA),
Safety, both)

\emph{Part 3: Elements scored regarding tumourigenicity studies}

Availability \emph{in vivo} study (no, yes); Reason for absence of the
study (not necessary, planned, requested, not necessary but
justification required, not mentioned); Species used (specified); Animal
model used (healthy, disease model, immune-compromised); Survival
product in animal (no, yes, not mentioned); Control group (no, yes, not
mentioned); Sample size (specified); Cell dose (specified); Route of
Administration (RoA, specified); Duration of the study (specified);
Tumor formation (no, yes of human origin, yes of animal origin,
unknown); ); Relevance (no, yes); Available \emph{In vitro} studies (no,
yes, requested, not mentioned); Method \emph{in vitro} study
(specified); \emph{In vivo} and \emph{In vitro} present (only \emph{in
vivo,} only \emph{in vitro} , both, neither performed, not mentioned);
Literature on cell type/cell product (no, yes, requested);
Informativeness total package (no, partially, yes, not discussed),
Reason for regarded informative (specified)

\emph{Analysis table}

\emph{Part 1: purpose of in vivo animal studies}

PoC/ MoA; Biodistribution; Tumourigenicity; Safety; Biodistribution \&
safety; Biodistribution \& POC; Tumourigenicity \& Safety;
Biodistribution \& tumourigenicity \& safety; Biodistribution \& PoC \&
safety; Bio \& POC \& safety; POC \& safety; POC \& tumourigenicity \&
safety; Biodistribution \& tumourigenicity

\emph{Part 2: need for, type and design of in vivo biodistribution
studies}

Available \emph{in vivo} study, Reason for absence (not necessary,
planned, requested, not necessary but justification required, not
mentioned); Multiple studies performed (no, yes); Homologous model (not
(known), yes, both homologous and heterologous); Migration (target
tissues, both target and non-target tissues); Quantifiable method (no,
yes, not mentioned); Persistence studied (no, yes, planned, not
mentioned); Informative (no, yes); Informative for (PoC/MoA, Safety,
both)

\emph{Part 3: need for, type and design of invitro and in vivo
tumourigenicity studies}

Available \emph{in vivo} study, Reason for absence (not necessary,
planned, requested, not necessary but justification required, not
mentioned); Homologous model (not (known), yes, both homologous and
heterologous); Dose of 10\^{}5; Dose of 10\^{}6; Dose of 10\^{}7, RoA
subcutaneous, RoA other (specified); Duration 0-3 months; Duration 3-6
months; Duration 6+ months; Presence Integration site analysis; Tumor
formation (no, yes of human origin, yes of animal origin, unknown);
Relevance (no, yes); Available \emph{In vitro} studies (no, yes,
requested, not mentioned); Karyotyping; Colony formation/Proliferation;
Genomic analysis (transcriptional/epigenetic/SNP/microarray, telomere
length); Immortalization (IVIM, senescence, apoptosis); Other
(specified); \emph{In vivo} and \emph{In vitro} present (only \emph{in
vivo,} only \emph{in vitro} , both, neither performed, not mentioned);
Informativeness total package (no, partially, yes, not discussed),
Reason for regarded informative (specified)\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
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\end{figure}\selectlanguage{english}
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\end{figure}\selectlanguage{english}
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\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
\end{figure}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}

\end{center}
\end{figure}

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