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@ -2373,6 +2373,58 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
publisher = {The American Association of Immunologists}, publisher = {The American Association of Immunologists},
} }
@Article{Kirouac2008,
author = {Daniel C. Kirouac and Peter W. Zandstra},
journal = {Cell Stem Cell},
title = {The Systematic Production of Cells for Cell Therapies},
year = {2008},
month = {oct},
number = {4},
pages = {369--381},
volume = {3},
doi = {10.1016/j.stem.2008.09.001},
publisher = {Elsevier {BV}},
}
@Article{Little2006,
author = {Melissa Little and Wayne Hall and Amy Orlandi},
journal = {{EMBO} reports},
title = {Delivering on the promise of human stem-cell research. What are the real barriers?},
year = {2006},
month = {nov},
number = {12},
pages = {1188--1192},
volume = {7},
doi = {10.1038/sj.embor.7400861},
publisher = {{EMBO}},
}
@Article{Pirnay2012,
author = {Jean-Paul Pirnay and Alain Vanderkelen and Nadine Ectors and Christian Delloye and Denis Dufrane and Etienne Baudoux and Michel Van Brussel and Michael P. Casaer and Daniel De Vos and Jean-Pierre Draye and Thomas Rose and Serge Jennes and Pierre Neirinckx and Geert Laire and Martin Zizi and Gilbert Verbeken},
journal = {Cell and Tissue Banking},
title = {Beware of the commercialization of human cells and tissues: situation in the European Union},
year = {2012},
month = {jun},
number = {3},
pages = {487--498},
volume = {13},
doi = {10.1007/s10561-012-9323-3},
publisher = {Springer Science and Business Media {LLC}},
}
@Article{Rousseau2013,
author = {Guillaume F. Rousseau and Marie-Catherine Giarratana and Luc Douay},
journal = {Biotechnology Journal},
title = {Large-scale production of red blood cells from stem cells: What are the technical challenges ahead?},
year = {2013},
month = {oct},
number = {1},
pages = {28--38},
volume = {9},
doi = {10.1002/biot.201200368},
publisher = {Wiley},
}
@Comment{jabref-meta: databaseType:bibtex;} @Comment{jabref-meta: databaseType:bibtex;}
@Comment{jabref-meta: grouping: @Comment{jabref-meta: grouping:

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tex/thesis.tex
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@ -158,6 +158,7 @@
\newacronym{hla}{HLA}{human leukocyte antigen} \newacronym{hla}{HLA}{human leukocyte antigen}
\newacronym{zfn}{ZFN}{zinc-finger nuclease} \newacronym{zfn}{ZFN}{zinc-finger nuclease}
\newacronym{talen}{TALEN}{transcription activator-like effector nuclease} \newacronym{talen}{TALEN}{transcription activator-like effector nuclease}
\newacronym{qbd}{QbD}{quality-by-design}
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% SI units for uber nerds % SI units for uber nerds
@ -571,12 +572,150 @@ present our final conclusions in Chapter~\ref{conclusions}.
\chapter{background and significance}\label{background} \chapter{background and significance}\label{background}
\section*{background} \section*{background}
% TODO mention cloudz stuff \subsection{quality by design in cell manufacturing}
% TODO break this apart into mfg tech and T cell phenotypes/quality The challenges facing the cell manufacturing field at large are significant.
% TODO consider adding a separate section on microcarriers and their use in Unlike other industries which manufacture inanimate products such as automobiles
% bioprocess and semiconductors, the cell manufacturing industry needs to contend with the
% TODO add stuff about T cell licensing fact that cells are living entities which can change with every process
manipulation\cite{Kirouac2008, Little2006, Pirnay2012, Rousseau2013}. This is
further compounded by the lack of standardization and limited regulation.
In order to overcome these barriers, adopting a systemic approach to cell
manufacturing using \gls{qbd} principles will be extremely
important\cite{Kirouac2008}. In \gls{qbd}, the objective is to reproducibly
manufacturing products which minimizes risk for downstream
stakeholders (in this case, the patient).
This requires identification of \glspl{cqa}, which are measurable properties of
the product that can be used to define its functionality and hence quality.
\glspl{cqa} are important for defining the characteristics of a `good' product
(release criteria) but also for ensuring that a process is on track to making
such a product (process control). In the space of cell manufacturing,
examples of \glspl{cqa} include markers on the surface of cells and readouts
from functional assays such as killing assays. In general, these are poorly
understood if they exist at all.
In addition to \glspl{cqa}, the \glspl{cpp} pertinent to the manufacturing
process are poorly understood. \glspl{cpp} are parameters which may be tuned and
varied to control the outcome of process and the quality of the final product.
Examples in the cell manufacturing space include the type of media used and the
amount of \il{2} added. Once \glspl{cpp} are known, they can be optimized to
ensure that costs are minimized and potency of the cellular product is
maximized.
The topic of discovering novel \glspl{cpp} and \glspl{cqa} in the context of
this work are discussed further in \cref{sec:background_doe} and
\cref{sec:background_quality}/\cref{sec:background_cqa} respectively.
\subsection{T cells for immunotherapies}
One of the first successful T cell-based immunotherapies against cancer is
\glspl{til}\cite{Rosenberg2015}. This method works by taking tumor specimens
from a patient, allowing the tumor-reactive lymphocytes to expand \exvivo{}, and
then administered back to the patient along with a high dose of \il{2} [44]. In
particular, \gls{til} therapy has shown robust results in treating melanoma [1],
although \gls{til} have been found in other solid tumors such as
gastointestinal, cervical, lung, and ovarian\cite{Rosenberg2015, Wang2014,
Foppen2015, Solinas2017, June2007, Santoiemma2015}, and their presence is
generally associate with favorable outcomes\cite{Clark1989}. \glspl{til} are
heterogenous cell mixtures and generally are comprised of CD3 T cells and
$\upgamma\updelta$ T cells\cite{Nishimura1999, Cordova2012}. To date, there are
over 250 open clinical trials using \glspl{til}.
Besides \gls{til}, the other broad class of T cell immunotherapies that has
achieved great success in treating cancer in recent decades are gene-modified T
cells. Rather than expand T cells that are present natively (as is the case with
\gls{til} therapy), gene-modified T cell therapies entail extracting T cells
from either the cancer patient (autologous) or a healthy donor (allogeneic) and
reprogramming them genetically to target a tumor antigen (see
\cref{sec:background_source}). In theory this offers much more
flexibility\cite{Rosenberg2015}.
T cells with transduced \glspl{tcr} were first designed to overcome the
limitations of \gls{til}\cite{Rosenberg2015, Wang2014}. In this case, T cells
are transduced \exvivo{} with a lentiviral vector to express a \gls{tcr}
targeting a tumor antigen. T cells transduced with \glspl{tcr} have shown robust
results in melanoma patients\cite{Robbins2011}, synovial
sarcoma\cite{Morgan2006}, and others\cite{Ikeda2016}. To date, there are over
200 clinical trials using T cells with transduced \glspl{tcr}.
While transduced \glspl{tcr} offer some flexibility in retargeting T cells
toward relevant tumor antigens, they are still limited in that they can only
target antigens that are presented via \gls{mhc} complexes. \gls{car} T cells
overcome this limitation by using a the heavy and light chains (scFv) from a
\gls{mab} which can target any antigen recognizable by antibodies. \gls{car} T
cells were first demonstrated in 1989, where the author swapped the
antigen-recognition domains of a native \gls{tcr} with a that of a foreign
\gls{tcr}\cite{Gross1989}. Since then, this method has progressed to using an
scFv with a CD3$\upzeta$ stimulatory domain along with the CD28, OX-40, or 4-1BB
domains for costimulation. Since these can all be expressed with one protein
sequence, \gls{car} T cells are relatively simple to produce and require only a
single genetic transduction step (usually a lentiviral vector) to reprogram a
batch T cells \exvivo{} toward the desired antigen. \gls{car} T cells have
primarily found success in against CD19- and CD20-expressing tumors such as
\gls{all} and \gls{cll} (eg B-cell malignancies)\cite{Kalos2011, Brentjens2011,
Kochenderfer2010, Maude2014, Till2012, Till2008}.
% BACKGROUND where else have they been approved?
Out of all the T cell therapies discussed thus far, \gls{car} T cells have
experienced the most commercial success and excitement. In 2017, Novartis and
Kite Pharma acquired FDA approval for \textit{Kymriah} and \textit{Yescarta}
respectively, both of which are \gls{car} T cell therapies against B-cell
malignancies.
% BACKGROUND beef this up, this is a big deal
\gls{car} T cells are under further exploration for use in many other tumors,
including multiple myeloma, mesothelioma, pancreatic cancer, glioblastoma,
neuroblastoma, and prostate cancer, breast cancer, non-small-cell lung cancer,
and others\cite{Rosenberg2015, Wang2014, Fesnak2016, Guo2016}. To date, there
are almost 1000 clinical trials using \gls{car} T cells.
% TODO there are other T cells like virus-specific T cells and gd T cells, not
% that they matter...
\subsection{cell sources in T cell manufacturing}\label{sec:background_source}
T cells for cell manufacturing can be obtained broadly via two paradigms:
autologous and allogeneic. The former involves obtaining T cells from a patient
and giving them back to the same patient after \exvivo{} expansion and genetic
modification. The latter involves taking T cells from a (usually) healthy donor,
expanding them and manipulating them as desired, storing them long term, and
then giving them to multiple patients. There are advantages and disadvantages to
both, and in some cases such as \gls{til} therapy, the only option is to use
autologous therapy.
Autologous T cells by default are much safer. By definition, they will have no
cross-reactivity with the patient and thus \gls{gvhd} is not a
concern\cite{Decker2012}. However, there are numerous disadvantages. Autologous
therapies are over 20X more costly as the process needs to be repeated for every
patient\cite{Harrison2019}. To highlight how resource-intensive this can be,
many cell products are manufactured at a centralized location, so patient T
cells need to be shipped twice on dry ice from the hospital and back. In
additional to being expensive, this can add days to the process, which is
critical for patients with fast moving diseases. Manufacturing could be done
on-site in a decentralized manner, but this requires more equipment and
personnel overall. Using cells from a diseased patient has many drawbacks in
itself. Cancer patients (especially those with chronic illnesses) often have
exhausted T cells which expand far less readily and are consequently less
potent\cite{Wherry2015, Ando2019, Zheng2017}. Additionally, they may have high
frequencies of T\textsubscript{reg} cells which inhibitory\cite{Decker2012}.
Removing these cells as well as purifying Th1 cells may enhance the potency of
the final product\cite{Goldstein2012, Drela2004, Rankin2003, Luheshi2013,
Grotz2015}; however, this would make the overall process more expensive as an
additional step would be required.
Allogeneic T cell therapies overcome nearly all of these disadvantages. Donor
\glspl{pbmc} are easy to obtain, they can be processed in centralized locations,
they can be stored easily under liquid nitrogen, and donors could be screened to
find those with optimal anti-tumor cells. The key is overcoming \gls{gvhd}.
Obviously this could be done the same way as done for transplants where patients
find a `match' for their \gls{hla} type, but this severally limits options. This
can be overcome by using gene-editing (eg \glspl{zfn}, \glspl{talen}, or
\gls{crispr} to remove the native \gls{tcr} which would prevent the donor T
cells from attacking host tissue\cite{Liu2019, Wiebking2020, Provasi2012,
Berdien2014, Themeli2015}. To date there are about 10 open clinical trials
utilizing allogeneic T cell therapies edited with \gls{crispr} to reduce the
likelihood of \gls{gvhd}.
\subsection{microcarriers in bioprocessing} \subsection{microcarriers in bioprocessing}
@ -634,114 +773,6 @@ therapies in the context of regenerative medicine\cite{Zhang2016, Saltz2016,
in this application is the fact that they are adherent. In this work, we explore in this application is the fact that they are adherent. In this work, we explore
the use of microcarrier for T cells, which are naturally non-adherent. the use of microcarrier for T cells, which are naturally non-adherent.
\subsection{overview of T cells in immunotherapies}
One of the first successful T cell-based immunotherapies against cancer is
\glspl{til}\cite{Rosenberg2015}. This method works by taking tumor specimens
from a patient, allowing the tumor-reactive lymphocytes to expand \exvivo{}, and
then administered back to the patient along with a high dose of \il{2} [44]. In
particular, \gls{til} therapy has shown robust results in treating melanoma [1],
although \gls{til} have been found in other solid tumors such as
gastointestinal, cervical, lung, and ovarian\cite{Rosenberg2015, Wang2014,
Foppen2015, Solinas2017, June2007, Santoiemma2015}, and their presence is
generally associate with favorable outcomes\cite{Clark1989}. \glspl{til} are
heterogenous cell mixtures and generally are comprised of CD3 T cells and
$\upgamma\updelta$ T cells\cite{Nishimura1999, Cordova2012}. To date, there are
over 250 open clinical trials using \glspl{til}.
Besides \gls{til}, the other broad class of T cell immunotherapies that has
achieved great success in treating cancer in recent decades are gene-modified T
cells. Rather than expand T cells that are present natively (as is the case with
\gls{til} therapy), gene-modified T cell therapies entail extracting T cells
from either the cancer patient (autologous) or a healthy donor (allogeneic) and
reprogramming them genetically to target a tumor antigen. In theory this offers
much more flexibility\cite{Rosenberg2015}.
T cells with transduced \glspl{tcr} were first designed to overcome the
limitations of \gls{til}\cite{Rosenberg2015, Wang2014}. In this case, T cells
are transduced \exvivo{} with a lentiviral vector to express a \gls{tcr}
targeting a tumor antigen. T cells transduced with \glspl{tcr} have shown robust
results in melanoma patients\cite{Robbins2011}, synovial
sarcoma\cite{Morgan2006}, and others\cite{Ikeda2016}. To date, there are over
200 clinical trials using T cells with transduced \glspl{tcr}.
While transduced \glspl{tcr} offer some flexibility in retargeting T cells
toward relevant tumor antigens, they are still limited in that they can only
target antigens that are presented via \gls{mhc} complexes. \gls{car} T cells
overcome this limitation by using a the heavy and light chains (scFv) from a
\gls{mab} which can target any antigen recognizable by antibodies. \gls{car} T
cells were first demonstrated in 1989, where the author swapped the
antigen-recognition domains of a native \gls{tcr} with a that of a foreign
\gls{tcr}\cite{Gross1989}. Since then, this method has progressed to using an
scFv with a CD3$\upzeta$ stimulatory domain along with the CD28, OX-40, or 4-1BB
domains for costimulation. Since these can all be expressed with one protein
sequence, \gls{car} T cells are relatively simple to produce and require only a
single genetic transduction step (usually a lentiviral vector) to reprogram a
batch T cells \exvivo{} toward the desired antigen. \gls{car} T cells have
primarily found success in against CD19- and CD20-expressing tumors such as
\gls{all} and \gls{cll} (eg B-cell malignancies)\cite{Kalos2011, Brentjens2011,
Kochenderfer2010, Maude2014, Till2012, Till2008}.
% BACKGROUND where else have they been approved?
Out of all the T cell therapies discussed thus far, \gls{car} T cells have
experienced the most commercial success and excitement. In 2017, Novartis and
Kite Pharma acquired FDA approval for \textit{Kymriah} and \textit{Yescarta}
respectively, both of which are \gls{car} T cell therapies against B-cell
malignancies.
% BACKGROUND beef this up, this is a big deal
\gls{car} T cells are under further exploration for use in many other tumors,
including multiple myeloma, mesothelioma, pancreatic cancer, glioblastoma,
neuroblastoma, and prostate cancer, breast cancer, non-small-cell lung cancer,
and others\cite{Rosenberg2015, Wang2014, Fesnak2016, Guo2016}. To date, there
are almost 1000 clinical trials using \gls{car} T cells.
% TODO there are other T cells like virus-specific T cells and gd T cells, not
% that they matter...
\subsection{cell sources in T cell manufacturing}
T cells for cell manufacturing can be obtained broadly via two paradigms:
autologous and allogeneic. The former involves obtaining T cells from a patient
and giving them back to the same patient after \exvivo{} expansion and genetic
modification. The latter involves taking T cells from a (usually) healthy donor,
expanding them and manipulating them as desired, storing them long term, and
then giving them to multiple patients. There are advantages and disadvantages to
both, and in some cases such as \gls{til} therapy, the only option is to use
autologous therapy.
Autologous T cells by default are much safer. By definition, they will have no
cross-reactivity with the patient and thus \gls{gvhd} is not a
concern\cite{Decker2012}. However, there are numerous disadvantages. Autologous
therapies are over 20X more costly as the process needs to be repeated for every
patient\cite{Harrison2019}. To highlight how resource-intensive this can be,
many cell products are manufactured at a centralized location, so patient T
cells need to be shipped twice on dry ice from the hospital and back. In
additional to being expensive, this can add days to the process, which is
critical for patients with fast moving diseases. Manufacturing could be done
on-site in a decentralized manner, but this requires more equipment and
personnel overall. Using cells from a diseased patient has many drawbacks in
itself. Cancer patients (especially those with chronic illnesses) often have
exhausted T cells which expand far less readily and are consequently less
potent\cite{Wherry2015, Ando2019, Zheng2017}. Additionally, they may have high
frequencies of T\textsubscript{reg} cells which inhibitory\cite{Decker2012}.
Removing these cells as well as purifying Th1 cells may enhance the potency of
the final product\cite{Goldstein2012, Drela2004, Rankin2003, Luheshi2013,
Grotz2015}; however, this would make the overall process more expensive as an
additional step would be required.
Allogeneic T cell therapies overcome nearly all of these disadvantages. Donor
\glspl{pbmc} are easy to obtain, they can be processed in centralized locations,
they can be stored easily under liquid nitrogen, and donors could be screened to
find those with optimal anti-tumor cells. The key is overcoming \gls{gvhd}.
Obviously this could be done the same way as done for transplants where patients
find a `match' for their \gls{hla} type, but this severally limits options. This
can be overcome by using gene-editing (eg \glspl{zfn}, \glspl{talen}, or
\gls{crispr} to remove the native \gls{tcr} which would prevent the donor T
cells from attacking host tissue\cite{Liu2019, Wiebking2020, Provasi2012,
Berdien2014, Themeli2015}. To date there are about 10 open clinical trials
utilizing allogeneic T cell therapies edited with \gls{crispr} to reduce the
likelihood of \gls{gvhd}.
\subsection{methods to scale T cells} \subsection{methods to scale T cells}
In order to scale T cell therapies to meet clinical demands, automation and In order to scale T cell therapies to meet clinical demands, automation and
@ -940,8 +971,8 @@ against Fas-mediated apoptosis in the presence of collagen I\cite{Aoudjit2000,
production \invitro{} when T cells derived from human \glspl{pbmc} are production \invitro{} when T cells derived from human \glspl{pbmc} are
stimulated in the presence of collagen I\cite{Boisvert2007}. stimulated in the presence of collagen I\cite{Boisvert2007}.
% TODO there are other receptors I could name here that were not explored % TODO there are other receptors I could name here that were not explored Other
% Other integrins that interact with the environment include a4b1, which interacts % integrins that interact with the environment include a4b1, which interacts
% with fibronectin and has been shown to lead to higher IL2 production (Iwata et % with fibronectin and has been shown to lead to higher IL2 production (Iwata et
% al 2000). % al 2000).
@ -996,7 +1027,7 @@ a soluble form, which can bind to \il{15} and signal to cells which are not
adjacent to the source independent of the \textit{cis/trans} mechanisms already adjacent to the source independent of the \textit{cis/trans} mechanisms already
described\cite{Budagian2004}. described\cite{Budagian2004}.
\subsection*{overview of design of experiments} \subsection*{overview of design of experiments}\label{sec:background_doe}
The \gls{dms} system has a number of parameters that can be optimized, and a The \gls{dms} system has a number of parameters that can be optimized, and a
\gls{doe} is an ideal framework to test multiple parameters simultaneously. The \gls{doe} is an ideal framework to test multiple parameters simultaneously. The
@ -1072,7 +1103,10 @@ directions for future work. To this end, the types of \glspl{doe} we generally
used in this work were fractional factorial designs with three levels, which used in this work were fractional factorial designs with three levels, which
enable the estimation of both main effects and second order quadratic effects. enable the estimation of both main effects and second order quadratic effects.
\subsection*{strategies to characterize cell manufacturing} \subsection*{identification and standardization of CPPs and CQAs}\label{sec:background_cqa}
Ultimately the identification of relevant \glspl{cpp} and \glspl{cqa} is an
interative process
A number of multiomics strategies exist which can generate rich datasets for T A number of multiomics strategies exist which can generate rich datasets for T
cells. We will consider several multiomics strategies within this proposal: cells. We will consider several multiomics strategies within this proposal: