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@ -581,14 +581,14 @@ modern manufacturing process.
The goal of this dissertation was to develop a microcarrier-based \gls{dms} T The goal of this dissertation was to develop a microcarrier-based \gls{dms} T
cell expansion system and determine biologically-meaningful \glspl{cqa} and cell expansion system and determine biologically-meaningful \glspl{cqa} and
\glspl{cpp} that could be used to optimize for highly-potent T cells. In \glspl{cpp} that could be used to optimize for highly-potent T cells. In
\cref{aim1}, we develop and characterized the \gls{dms} system, including \cref{aim1}, we developed and characterized the \gls{dms} system, including
quality control steps. We also demonstrate the feasibility of expanding quality control steps. We also demonstrated the feasibility of expanding
high-quality T cells. In \cref{aim2a,aim2b}, we use \gls{doe} methodology to high-quality T cells. In \cref{aim2a,aim2b}, we used \gls{doe} methodology to
optimize the \gls{dms} platform, and we develop a computational pipeline to optimize the \gls{dms} platform, and we developed a computational pipeline to
identify and model the effects of measurable \glspl{cqa} and \glspl{cpp} on the identify and model the effects of measurable \glspl{cqa} and \glspl{cpp} on the
final product. In \cref{aim3}, we demonstrate the effectiveness of the \gls{dms} final product. In \cref{aim3}, we demonstrated the effectiveness of the
platform \invivo{}. This thesis lays the groundwork for a novel T cell expansion \gls{dms} platform \invivo{}. This thesis lays the groundwork for a novel T cell
method which can be utilized at scale for clinical trials and beyond. expansion method which can be utilized at scale for clinical trials and beyond.
\clearpage \clearpage
@ -604,39 +604,39 @@ diseases\cite{Fesnak2016,Rosenberg2015}. In 2017, Novartis and Kite Pharma
received FDA approval for \textit{Kymriah} and \textit{Yescarta} respectively, received FDA approval for \textit{Kymriah} and \textit{Yescarta} respectively,
two genetically-modified \gls{car} T cell therapies against B cell malignancies. two genetically-modified \gls{car} T cell therapies against B cell malignancies.
Despite these successes, \gls{car} T cell therapies are constrained by an Despite these successes, \gls{car} T cell therapies are constrained by an
expensive and difficult-to-scale manufacturing process with little control on expensive, difficult-to-scale manufacturing process with little control on cell
cell quality and phenotype\cite{Roddie2019, Dwarshuis2017}. State-of-the-art T quality and phenotype\cite{Roddie2019, Dwarshuis2017}. State-of-the-art T cell
cell manufacturing techniques focus on \acd{3} and \acd{28} activation and manufacturing techniques focus on \acd{3} and \acd{28} activation and expansion,
expansion, typically presented on superparamagnetic, iron-based microbeads typically presented on superparamagnetic, iron-based microbeads (Invitrogen
(Invitrogen Dynabead, Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or Dynabead, Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or in soluble
in soluble tetramers (Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, tetramers (Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017,
Piscopo2017, Bashour2015}. These strategies overlook many of the signaling Bashour2015}. These strategies overlook many of the signaling components
components present in the secondary lymphoid organs where T cells expand present in the secondary lymphoid organs where T cells expand \invivo{}.
\invivo{}. Typically, T cells are activated under close cell-cell contact, which Typically, T cells are activated under close cell-cell contact, which allows for
allows for efficient autocrine/paracrine signaling via growth-stimulating efficient autocrine/paracrine signaling via growth-stimulating cytokines such as
cytokines such as \gls{il2}. Additionally, the lymphoid tissues are comprised of \gls{il2}. Additionally, the lymphoid tissues are comprised of \gls{ecm}
\gls{ecm} components such as collagen and stromal cells, which provide signals components such as collagen and stromal cells, which provide signals to
to upregulate proliferation, cytokine production, and pro-survival upregulate proliferation, cytokine production, and pro-survival
pathways\cite{Gendron2003, Ohtani2008, Boisvert2007, Ben-Horin2004}. pathways\cite{Gendron2003, Ohtani2008, Boisvert2007, Ben-Horin2004}.
A variety of solutions have been proposed to make the T cell expansion process A variety of solutions have been proposed to make the T cell expansion process
more physiological. Including feeder cell cultures\cite{Forget2014} and more physiological. These include feeder cell cultures\cite{Forget2014} and
biomaterials-based methods such as lipid-coated microrods or 3D scaffold biomaterials-based methods such as lipid-coated microrods or 3D scaffold
gels\cite{Cheung2018,Delalat2017,meyer15_immun,Lambert2017} that attempt to gels\cite{Cheung2018,Delalat2017,meyer15_immun,Lambert2017} that attempt to
recapitulate the cellular membrane, large interfacial contact area, recapitulate the cellular membrane, large interfacial contact area,
3D-structure, or soft surfaces T cells normally experience \invivo{}. While 3D-structure, or soft surfaces T cells normally experience \invivo{}. While
these have been shown to activation and expand T cells, they either are not these have been shown to activate and expand T cells, they either are not
scalable (in the case of feeder cells) or still lack many of the signals and scalable (in the case of feeder cells) or still lack many of the signals and
cues T cells experience as the expand. Additionally, none have been shown to cues T cells experience as the expand. Additionally, none have been shown to
preferentially expand highly-potent T cell necessary for anti-cancer therapies. preferentially expand highly-potent T cell necessary for anti-cancer therapies.
Such high potency cells including subtypes with low differentiation state such Such high potency cells are subtypes with low differentiation state such
as \gls{tscm} and \gls{tcm} cells or CD4 cells, all of which have been shown to as \gls{tscm} and \gls{tcm} cells or CD4 cells, all of which have been shown to
be necessary for durable responses\cite{Xu2014, Fraietta2018, Gattinoni2011, be necessary for durable responses\cite{Xu2014, Fraietta2018, Gattinoni2011,
Gattinoni2012,Wang2018, Yang2017}. Methods to increase memory and CD4 T cells Gattinoni2012,Wang2018, Yang2017}. Methods to increase memory and CD4 T cells
in the final product are needed. Furthermore, \gls{qbd} principles such as in the final product are needed. Furthermore, \gls{qbd} principles such as
discovering and validating novel \glspl{cqa} and \glspl{cpp} in the space of T discovering and validating novel \glspl{cqa} and \glspl{cpp} in the space of T
cell manufacturing are required to reproducibly manufacture these subtypes and cell manufacturing are required to reproducibly manufacture these subtypes and
ensure low-cost and safe products with maximal effectiveness in the clinic ensure low-cost and safe products with maximal effectiveness in the clinic.
This dissertation describes a novel \acrlong{dms}-based method derived from This dissertation describes a novel \acrlong{dms}-based method derived from
porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} for porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} for
@ -654,11 +654,11 @@ emulate the large contact surface area that occurs between T cells and
\section*{hypothesis} \section*{hypothesis}
The hypothesis of this dissertation was that using \glspl{dms} created from The hypothesis of this dissertation was that using \glspl{dms} created from
off-the-shelf microcarriers and coated with activating \glspl{mab} would lead to off-the-shelf microcarriers and coated with activating \glspl{mab} would
higher quantity and quality T cells as compared to state-of-the-art bead-based increase quantity and quality of T cells as compared to state-of-the-art
expansion. We also hypothesized that T cells have measurable biological bead-based expansion. We also hypothesized that such T cells have measurable
signatures that are predictive of downstream outcomes and phenotypes. The biological signatures that are predictive of downstream outcomes and phenotypes.
objective of this dissertation was to develop this platform, test its The objective of this dissertation was to develop this platform, test its
effectiveness both \invitro{} and \invivo{}, and develop computational pipelines effectiveness both \invitro{} and \invivo{}, and develop computational pipelines
to discover novel \glspl{cpp} and \glspl{cqa} that can be translated to a to discover novel \glspl{cpp} and \glspl{cqa} that can be translated to a
manufacturing environment and a clinical trial setting. manufacturing environment and a clinical trial setting.
@ -683,10 +683,10 @@ The specific aims of this dissertation are outlined in
In this first aim, we demonstrated the process for manufacturing \glspl{dms}, In this first aim, we demonstrated the process for manufacturing \glspl{dms},
including quality control steps that are necessary for translation of this including quality control steps that are necessary for translation of this
platform into a scalable manufacturing setting. We also demonstrate that the platform into a scalable manufacturing setting. We also demonstrated that the
\gls{dms} platform leads to higher overall expansion of T cells and higher \gls{dms} platform leads to higher overall expansion of T cells and higher
overall fractions of potent memory and CD4+ subtypes desired for T cell overall fractions of potent memory and CD4+ subtypes desired for T cell
therapies. Finally, we demonstrate \invitro{} that the \gls{dms} platform can be therapies. Finally, we showed \invitro{} that the \gls{dms} platform can be
used to generate functional \gls{car} T cells targeted toward CD19. used to generate functional \gls{car} T cells targeted toward CD19.
\subsection*{aim 2: develop methods to control and predict T cell quality} \subsection*{aim 2: develop methods to control and predict T cell quality}
@ -705,7 +705,7 @@ accomplished through two sub-aims:
\subsection*{aim 3: confirm potency of T cells from novel T cell expansion \subsection*{aim 3: confirm potency of T cells from novel T cell expansion
process using \invivo{} xenograft mouse model} process using \invivo{} xenograft mouse model}
In this final aim, we demonstrate the effectiveness of \gls{dms}-expanded T In this final aim, we demonstrated the effectiveness of \gls{dms}-expanded T
cells compared to state-of-the-art beads using \invivo{} mouse models for cells compared to state-of-the-art beads using \invivo{} mouse models for
\gls{all}. \gls{all}.
@ -732,27 +732,28 @@ manipulation\cite{Kirouac2008, Little2006, Pirnay2012, Rousseau2013}. This is
further compounded by the lack of standardization and limited regulation. further compounded by the lack of standardization and limited regulation.
In order to overcome these barriers, adopting a systemic approach to cell In order to overcome these barriers, adopting a systemic approach to cell
manufacturing using \acrlong{qbd} principles will be extremely manufacturing using \acrfull{qbd} principles will be extremely
important\cite{Kirouac2008}. In \gls{qbd}, the objective is to reproducibly important\cite{Kirouac2008}. In \gls{qbd}, the objective is to reproducibly
manufacturing products which minimizes risk for downstream stakeholders (in this manufacturing products which minimizes risk for downstream stakeholders (in this
case, the patient). Broadly, this entails determining \acrlongpl{cqa} and case, the patient). This entails determining \acrlongpl{cqa} and \acrlongpl{cpp}
\acrlongpl{cpp} and incorporating them into models which can explain and predict and incorporating them into models which can explain and predict the cell
the cell manufacturing process. manufacturing process.
\Glspl{cqa} are measurable properties of the product that can be used to define \Glspl{cqa} are measurable properties of the product that are used to define its
its functionality and hence quality. \glspl{cqa} are important for defining the functionality and hence quality. \glspl{cqa} are important for defining the
characteristics of a `good' product (release criteria) but also for ensuring 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 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 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 surface of cells and readouts from functional assays such as killing assays. In
general, these are poorly understood if they exist at all. general, these are poorly understood if they exist at all.
%% TODO IL2 use here is wonky
\glspl{cpp} are parameters which may be tuned and varied to control the outcome \glspl{cpp} are parameters which may be tuned and varied to control the outcome
of process and the quality of the final product. In cell manufacturing, these of process and the quality of the final product. Examples include the type of
are poorly understood. Examples in the cell manufacturing space include the type media used and the amount of \il{2} added. While these can be easy to control,
of media used and the amount of \il{2} added. Once \glspl{cpp} are known, they the effect they have on the final outcome is generally unknown. Once \glspl{cpp}
can be optimized to ensure that costs are minimized and potency of the cellular are known, they can be optimized to ensure that costs are minimized and potency
product is maximized. of the cellular product is maximized.
The topic of discovering novel \glspl{cpp} and \glspl{cqa} in the context of 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 this work are discussed further in \cref{sec:background_doe} and
@ -767,12 +768,13 @@ theory the technology developed in this dissertation could theoretically apply
to any T cell-based therapy with little to no modification. to any T cell-based therapy with little to no modification.
One of the first successful T cell-based immunotherapies against cancer is One of the first successful T cell-based immunotherapies against cancer is
\glspl{til}\cite{Rosenberg2015}. This method works by taking tumor specimens \glspl{til}\cite{Rosenberg2015}. This method works by excising tumor fragments
from a patient, allowing the tumor-reactive lymphocytes to expand \exvivo{}, and from a patient, allowing the tumor-reactive lymphocytes to expand \exvivo{} from
then administered back to the patient along with a high dose of within these fragments, and then administered these lymphocytes back to the
\il{2}\cite{Rosenberg1988}. In particular, \gls{til} therapy has shown robust patient along with a high dose of \il{2}\cite{Rosenberg1988}. In particular,
results in treating melanoma\cite{Rosenberg2011}, although \glspl{til} have been \gls{til} therapy has shown robust results in treating
found in other solid tumors such as gastointestinal, cervical, lung, and melanoma\cite{Rosenberg2011}, although \glspl{til} have been found in other
solid tumors such as gastointestinal, cervical, lung, and
ovarian\cite{Rosenberg2015, Wang2014, Foppen2015, Solinas2017, June2007, ovarian\cite{Rosenberg2015, Wang2014, Foppen2015, Solinas2017, June2007,
Santoiemma2015}, and their presence is generally associate with favorable Santoiemma2015}, and their presence is generally associate with favorable
outcomes\cite{Clark1989}. \glspl{til} are heterogeneous cell mixtures and outcomes\cite{Clark1989}. \glspl{til} are heterogeneous cell mixtures and
@ -795,26 +797,27 @@ T cells with transduced \glspl{tcr} were first designed to overcome the
limitations of \glspl{til}\cite{Rosenberg2015, Wang2014}. In this case, T cells limitations of \glspl{til}\cite{Rosenberg2015, Wang2014}. In this case, T cells
are transduced \exvivo{} with a lentiviral vector to express a \gls{tcr} 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 targeting a tumor antigen. T cells transduced with \glspl{tcr} have shown robust
results in melanoma patients\cite{Robbins2011}, synovial results against melanoma \cite{Robbins2011}, synovial sarcoma\cite{Morgan2006},
sarcoma\cite{Morgan2006}, and others\cite{Ikeda2016}. To date, there are over and others\cite{Ikeda2016}. To date, there are over 200 clinical trials using T
200 clinical trials using T cells with transduced \glspl{tcr}. cells with transduced \glspl{tcr}.
While transduced \glspl{tcr} offer some flexibility in retargeting T cells While transduced \glspl{tcr} offer some flexibility in retargeting T cells
toward relevant tumor antigens, they are still limited in that they can only toward relevant tumor antigens, they are still limited in that they can only
target antigens that are presented via \gls{mhc} complexes. \acrlong{car} T target antigens that are presented via \gls{mhc}. \Acrlong{car} T cells overcome
cells overcome this limitation by using linking a \gls{tcr}-independent antigen this limitation by linking a \gls{tcr}-independent antigen recognition domain
recognition domain with the stimulatory and costimulatory machinery of a T cell with the stimulatory and costimulatory machinery of a T cell. \gls{car} T cells
\gls{car} T cells were first demonstrated in 1989, where the authors swapped the were first demonstrated in 1989, where the authors swapped the
antigen-recognition domains of a native \gls{tcr} with a that of a foreign 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 \gls{tcr}\cite{Gross1989}. Since then, this method has progressed to using an
\gls{scfv} with a CD3$\upzeta$ stimulatory domain along with the CD28, OX-40, or \gls{scfv} for antigen recognition, a CD3$\upzeta$ domain for the stimulatory
4-1BB domains for costimulation. Since these can all be expressed with one signal, and a CD28, OX-40, or 4-1BB domains for the costimulatory signal. Since
protein sequence, \gls{car} T cells are relatively simple to produce and require these can all be expressed with one protein sequence, \gls{car} T cells are
only a single genetic transduction step (usually a lentiviral vector) to relatively simple to produce and require only a single genetic transduction step
reprogram a batch T cells \exvivo{} toward the desired antigen. \gls{car} T (usually a lentiviral vector) to reprogram a batch T cells \exvivo{} toward the
cells have primarily found success in against CD19- and CD20-expressing tumors desired antigen. \gls{car} T cells have primarily found success in against CD19-
such as \gls{all} and \gls{cll} (eg B-cell malignancies)\cite{Kalos2011, and CD20-expressing tumors such as \gls{all} and \gls{cll} (eg B-cell
Brentjens2011, Kochenderfer2010, Maude2014, Till2012, Till2008}. malignancies)\cite{Kalos2011, Brentjens2011, Kochenderfer2010, Maude2014,
Till2012, Till2008}.
Out of all the T cell therapies discussed thus far, \gls{car} T cells have 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 experienced the most commercial success and excitement. In 2017, Novartis and
@ -828,9 +831,8 @@ date, there are almost 1000 clinical trials using \gls{car} T cells.
\subsection{Scaling T Cell Expansion} \subsection{Scaling T Cell Expansion}
In order to scale T cell therapies to meet clinical demands, automation and In order to scale T cell therapies, automation and bioreactors will be
bioreactors will be necessary. To this end, there are several choices that have necessary. To this end, several choices have found success in the clinic.
found success in the clinic.
The WAVE bioreactor (GE Healthcare) is the choice of expansion for many clinical The WAVE bioreactor (GE Healthcare) is the choice of expansion for many clinical
applications\cite{Brentjens2011, Hollyman2009, Brentjens2013}. It is part of a applications\cite{Brentjens2011, Hollyman2009, Brentjens2013}. It is part of a
@ -844,32 +846,30 @@ design, which could have negative impact on cross-talk, activation, and
growth\cite{Somerville2012}. growth\cite{Somerville2012}.
% BACKGROUND find clinical trials (if any) that use this % BACKGROUND find clinical trials (if any) that use this
Alternatively, the CliniMACS Prodigy (Miltenyi) is an all-in-one system that is Alternatively, the CliniMACS Prodigy (Miltenyi) is an all-in-one, fully-closed
a fully closed system that removes the need for expensive cleanrooms and system that removes the need for expensive cleanrooms and associated
associated personnel\cite{Kaiser2015, Bunos2015}. It contains modules to perform personnel\cite{Kaiser2015, Bunos2015}. It contains modules to perform
transduction, expansion, and washing. This setup also implies that fewer transduction, expansion, and washing. This setup is less prone to mistakes,
mistakes and handling errors will be made, since many of the steps are internal since most steps are internal to the machine. Initial investigations have shown
to the machine. Initial investigations have shown that it can yield T cells that it can yield T cells doses required for clinical use\cite{Zhu2018}. At the
doses required for clinical use\cite{Zhu2018}. At the time of writing, several time of writing, several clinical trial are underway which use the CliniMACS,
clinical trial are underway which use the CliniMACS, although mostly for although mostly for stem-cell based cell treatments.
stem-cell based cell treatments.
Finally, another option that has been investigated for T cell expansion is the Finally, another option that has been investigated for T cell expansion is the
Grex bioreactor (Wilson Wolf). This is effectively a tall tissue-culture plate Grex bioreactor (Wilson Wolf). This is effectively a tall tissue-culture plate
with a porous membrane at the bottom, which allows gas exchange to the active with a porous membrane at the bottom. This allows large volumes of media to be
cell culture at the bottom of the plate while permitting large volumes of media loaded without suffocating the cells, which can exchange gas through the
to be loaded on top without suffocating the cells. While this is quite similar membrane. While this is quite similar to plates and flasks normally used for
to plates and flasks normally used for small-scale research, the important small-scale research, the important difference is that its larger size requires
difference is that its larger size requires fewer interactions and keeps the fewer interactions and keeps the cells at a higher nutrient concentration for
cells at a higher nutrient concentration for longer periods of time. However, it longer periods of time. However, it is still a an open system and requires
is still a an open system and requires manual (by default) interaction from an manual (by default) interaction from an operator to load, feed, and harvest the
operator to load, feed, and harvest the cell product. Grex bioreactors have been cell product. Grex bioreactors have been using to grow \glspl{til}\cite{Jin2012}
using to grow \glspl{til}\cite{Jin2012} and virus-specific T and virus-specific T cells\cite{Gerdemann2011}.
cells\cite{Gerdemann2011}.
Much work is still required in the space of bioreactor design for T cell Much work is still required in the space of bioreactor design for T cell
manufacturing, but novel T cell expansion technologies such as that described in manufacturing, but novel T cell expansion technologies such as that described in
this work need to consider how it may be used at scale in such a system. this work need to consider how they may be used at scale in such a system.
\subsection{Cell Sources in T Cell Manufacturing}\label{sec:background_source} \subsection{Cell Sources in T Cell Manufacturing}\label{sec:background_source}
@ -887,35 +887,34 @@ cross-reactivity with the patient and thus \gls{gvhd} is not a
concern\cite{Decker2012}. However, there are numerous disadvantages. Autologous concern\cite{Decker2012}. However, there are numerous disadvantages. Autologous
therapies are over 20 times more costly as the process needs to be repeated for therapies are over 20 times more costly as the process needs to be repeated for
every patient\cite{Harrison2019}. Compounding this, many cell products are every patient\cite{Harrison2019}. Compounding this, many cell products are
manufactured at a centralized location, so patient T cells need to be shipped manufactured at a centralized location, so cells need to be shipped on dry ice
twice on dry ice from the hospital and back. This adds days to the process, from the hospital and back. This adds days to the process, which is critical for
which is critical for patients with fast moving diseases. Manufacturing could be patients with fast moving diseases. Manufacturing could be done on-site in a
done on-site in a decentralized manner, but this requires more equipment and decentralized manner, but this requires more equipment and personnel overall.
personnel overall. Using cells from a diseased patient has many drawbacks in Sourcing cells from a diseased patient has many drawbacks in itself. Cancer
itself. Cancer patients (especially those with chronic illnesses) often have patients (especially those with chronic illnesses) often have exhausted T cells
exhausted T cells which expand far less readily and are consequently less which expand far less readily and are consequently less potent\cite{Wherry2015,
potent\cite{Wherry2015, Ando2019, Zheng2017}. Additionally, they may have high Ando2019, Zheng2017}. Additionally, they may have high frequencies of
frequencies of \glspl{treg} which inhibitory\cite{Decker2012}. Removing these \glspl{treg} which have an inhibitory effect on
cells as well as purifying \glspl{th1} may enhance the potency of the final immunotherapies\cite{Decker2012}. Removing these cells as well as purifying
product\cite{Goldstein2012, Drela2004, Rankin2003, Luheshi2013, Grotz2015}; \glspl{th1} may enhance the potency of the final product\cite{Goldstein2012,
however, this would make the overall process more expensive as an additional Drela2004, Rankin2003, Luheshi2013, Grotz2015}; however, this makes the
separation step would be required. overall process more expensive as an additional separation step is required.
Allogeneic T cell therapies overcome nearly all of these disadvantages. Donor Allogeneic T cell therapies overcome nearly all of these disadvantages. Donor
\glspl{pbmc} are easy to obtain, they can be processed in centralized locations, \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 and they can be stored easily under liquid nitrogen. Donors can also be screened
find those with optimal anti-tumor cells. The key is overcoming \gls{gvhd}. 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 Obviously this could be done analogously to transplants where patients find a
find a `match' for their \gls{hla} type, but this severally limits options. This ``match'' for their \gls{hla} type, but this severally limits options. This can
can be overcome by using advanced gene-editing tools which can both add and be overcome by using advanced gene-editing tools (eg \glspl{zfn}, \glspl{talen},
delete genetic material (eg \glspl{zfn}, \glspl{talen}, or \gls{crispr}) to or \gls{crispr}) to remove the native \gls{tcr} and thus prevent the donor T
remove the native \gls{tcr} which would prevent the donor T cells from attacking cells from attacking host tissue\cite{Liu2019, Wiebking2020, Provasi2012,
host tissue\cite{Liu2019, Wiebking2020, Provasi2012, Berdien2014, Themeli2015}. Berdien2014, Themeli2015}. This obviously complicates the process, as
This obviously complicates the process, as additional edits besides the additional edits besides the insertion of the \gls{car} would be required, and
insertion of the \gls{car} would be required, and these technologies are not yet these technologies are not yet very efficient. To date there are about 10 open
very efficient. To date there are about 10 open clinical trials utilizing clinical trials utilizing allogeneic T cell therapies edited with \gls{crispr}
allogeneic T cell therapies edited with \gls{crispr} to reduce the likelihood of to reduce the likelihood of \gls{gvhd}.
\gls{gvhd}.
\subsection{Overview of T Cell Quality}\label{sec:background_quality} \subsection{Overview of T Cell Quality}\label{sec:background_quality}
@ -927,13 +926,13 @@ criteria, and initial cell source screening.
One of the most important dimensions of T cell quality is that of One of the most important dimensions of T cell quality is that of
differentiation. T cells begin their life in circulation (eg after they exit the differentiation. T cells begin their life in circulation (eg after they exit the
thymus) as \glspl{tn}. When they become activated in the secondary lymph node thymus) as \glspl{tn}. When they become activated in the secondary lymphoid
organs, they differentiate from \gls{tn} to \glspl{tscm}, \glspl{tcm}, organs, they differentiate from \gls{tn} to \glspl{tscm}, \glspl{tcm},
\glspl{tem}, and finally \glspl{teff}\cite{Gattinoni2012}. Subtypes earlier in \glspl{tem}, and finally \glspl{teff}\cite{Gattinoni2012}. Subtypes earlier in
this process are generally called `memory' or `memory-like' cells (eg \gls{tscm} this process are generally called ``memory'' or ``memory-like'' cells (eg
and \gls{tcm}), and have been shown to have increased potency toward a variety \gls{tscm} and \gls{tcm}), and have been shown to have increased potency toward
of tumors, presumably due to their higher capacity for self-renewal and a variety of tumors, presumably due to their higher capacity for self-renewal
replication, enhanced migratory capacity, and/or increased engraftment and replication, enhanced migratory capacity, and/or increased engraftment
potential\cite{Xu2014, Gattinoni2012, Fraietta2018, Gattinoni2011, Turtle2009}. potential\cite{Xu2014, Gattinoni2012, Fraietta2018, Gattinoni2011, Turtle2009}.
The capacity for self-renewal is especially important for T cells therapies, as The capacity for self-renewal is especially important for T cells therapies, as
evidenced by the fact that \gls{til} therapies with longer telomeres tend to evidenced by the fact that \gls{til} therapies with longer telomeres tend to
@ -951,7 +950,7 @@ In addition to memory, the other major axis by which T cells may be classified
is the CD4/CD8 ratio. \Glspl{th} are CD4+ are responsible for secreting is the CD4/CD8 ratio. \Glspl{th} are CD4+ are responsible for secreting
cytokines which coordinate the immune response while CD8+ \glspl{tc} are cytokines which coordinate the immune response while CD8+ \glspl{tc} are
responsible for killing tumors or infected cells using specialized lytic responsible for killing tumors or infected cells using specialized lytic
enzymes. Since \glspl{tc} actually perform the killing function, it seems enzymes. Since \glspl{tc} actually possess the killing function, it seems
intuitive that \glspl{tc} would be most important for anti-tumor intuitive that \glspl{tc} would be most important for anti-tumor
immunotherapies. However, in mouse models with glioblastoma, survival was immunotherapies. However, in mouse models with glioblastoma, survival was
negatively impacted when \glspl{th} were removed\cite{Wang2018}. Furthermore, negatively impacted when \glspl{th} were removed\cite{Wang2018}. Furthermore,
@ -969,10 +968,10 @@ radioactive tracer, by measuring the degranulation of the T cells themselves, or
by measuring a cytokine that is secreted upon T cell activation and killing such by measuring a cytokine that is secreted upon T cell activation and killing such
as \gls{ifng}. Furthermore, the viability of T cells may be assessed using a as \gls{ifng}. Furthermore, the viability of T cells may be assessed using a
number of methods, including exclusion dyes such as \gls{aopi} or a functional number of methods, including exclusion dyes such as \gls{aopi} or a functional
assay to detect metabolism. Finally, for the purposes of safety, T cell products assay to detect metabolism. Finally, for safety, retro- or lentivirally
using retro- or lentiviral vectors as their means of gene-editing must be tested transduced T cell products must be tested for replication competent
for replication competent vectors\cite{Wang2013} and for contamination via vectors\cite{Wang2013}, and all cell products in general should be tested for
bacteria or other pathogens. bacterial or fungal contamination.
\subsection{T Cell Activation Methods}\label{sec:background_activation} \subsection{T Cell Activation Methods}\label{sec:background_activation}
@ -983,24 +982,23 @@ present a peptide via \gls{mhc} that the T cell in question is able to
recognize. Signal 2 is administered via CD80 and CD86 which are also present on recognize. Signal 2 is administered via CD80 and CD86 which are also present on
\glspl{apc} and is necessary to prevent the T cell from becoming anergic. While \glspl{apc} and is necessary to prevent the T cell from becoming anergic. While
these two signal are the bare minimum to trigger a T cell to expand, there are these two signal are the bare minimum to trigger a T cell to expand, there are
many other potential signals present. T cells have many other costimulatory many other potential signals present. T cells have other receptors such as OX40,
receptors such as OX40, 4-1BB and ICOS which are costimulatory along with CD28, 4-1BB, and ICOS which are costimulatory along with CD28, and \glspl{apc} have
and \glspl{apc} have corresponding ligands for these depending on the nature of corresponding ligands for these depending on the nature of the pathogen they
the pathogen they have detected\cite{Azuma2019}. Furthermore, T cells exist in have detected\cite{Azuma2019}. Furthermore, T cells exist in high cell density
high cell density within the secondary lymphoid organs, which allows efficient within the secondary lymphoid organs, which allows efficient cytokine cross-talk
cytokine cross-talk in an autocrine and paracrine manner. These cytokines are in an autocrine and paracrine manner. These cytokines are responsible for
responsible for triggering proliferation (in the case of \il{2}) and subset triggering proliferation (in the case of \il{2}) and subset differentiation (in
differentiation (in the case of many others)\cite{Luckheeram2012}. By tuning the the case of many others)\cite{Luckheeram2012}. By tuning the signal strength and
signal strength and duration of Signal 1, Signal 2, the various costimulatory duration of Signal 1, Signal 2, the various costimulatory signals, and the
signals, and the cytokine milieu, a variety of T cell phenotypes can be cytokine milieu, a variety of T cell phenotypes can be actualized.
actualized.
\Invitro{}, T cells can be activated in a number of ways but the simplest and There are many ways to activate T cells \invitro{}, but the simplest and most
most common is to use \glspl{mab} that cross-link the CD3 and CD28 receptors, common is to use \glspl{mab} that cross-link CD3 and CD28, which supply Signal 1
which supply Signal 1 and Signal 2 without the need for antigen (which also and Signal 2 without the need for antigen (which also means all T cells are
means all T cells are activated and not just a few specific clones). Additional activated and not just a few specific clones). Additional signals may be
signals may be supplied in the form of cytokines (eg \il{2}, \il{7}, or \il{15}) supplied in the form of cytokines (eg \il{2}, \il{7}, or \il{15}) or feeder
or feeder cells\cite{Forget2014}. cells\cite{Forget2014}.
As this is a critical unit operation in the manufacturing of T cell therapies, a As this is a critical unit operation in the manufacturing of T cell therapies, a
number of commercial technologies exist to activate T cells\cite{Wang2016, number of commercial technologies exist to activate T cells\cite{Wang2016,
@ -1009,11 +1007,11 @@ number of commercial technologies exist to activate T cells\cite{Wang2016,
accomplished in a \gls{gmp} manner (at least from a reagents perspective) as accomplished in a \gls{gmp} manner (at least from a reagents perspective) as
soluble \gls{gmp}-grade \glspl{mab} are commericially available. A similar but soluble \gls{gmp}-grade \glspl{mab} are commericially available. A similar but
distinct method along these lines is to use multivalent activators such as distinct method along these lines is to use multivalent activators such as
ImmunoCult (StemCell Technologies) or Expamer (Juno Therapeutics) which may have ImmunoCult (StemCell Technologies) or Expamer (Juno Therapeutics) which have
increased cross-linking capacity compared to traditional \glspl{mab}. Beyond increased cross-linking capacity compared to traditional \glspl{mab}. Beyond
soluble protein, \glspl{mab} against CD3 and CD28 can be mounted on magnetic soluble protein, \glspl{mab} against CD3 and CD28 can be mounted on magnetic
microbeads (\SIrange{3}{5}{\um} in diameter) such as DynaBeads (Invitrogen) and microbeads (\SIrange{3}{5}{\um} in diameter) such as DynaBeads (Invitrogen) and
MACSbeads (\miltenyi{}), which are easier to separate using magnetic washing MACSbeads (\miltenyi{}), which are easy to separate using magnetic washing
plates. Magnetic nanobeads such as TransAct (\miltenyi{}) work by a similar plates. Magnetic nanobeads such as TransAct (\miltenyi{}) work by a similar
principle except they can be removed via centrifugation rather than a magnetic principle except they can be removed via centrifugation rather than a magnetic
washing plate. Cloudz (RnD Systems) are another bead-based T cell expansion washing plate. Cloudz (RnD Systems) are another bead-based T cell expansion
@ -1026,18 +1024,17 @@ Signal 1 and Signal 2 and ignore the many other physiological cues present in
the secondary lymphoid organs. A variety of novel T cell activation and the secondary lymphoid organs. A variety of novel T cell activation and
expansion solutions have been proposed to overcome this. One strategy is to use expansion solutions have been proposed to overcome this. One strategy is to use
modified feeder cell cultures to provide activation signals similar to those of modified feeder cell cultures to provide activation signals similar to those of
\glspl{dc}\cite{Forget2014}. While this has the theoretical capacity to mimic \glspl{dc}\cite{Forget2014}. While this can theoretically mimic many components
several key components of the lymph node, it is hard to reproduce on a large of the lymph node, it is hard to scale due to the complexity and inherent
scale due to the complexity and inherent variability of using cell lines in a variability of using cell lines in a \gls{gmp}-compliant manner. Others have
fully \gls{gmp}-compliant manner. Others have proposed biomaterials-based proposed biomaterials-based solutions to circumvent this problem, including
solutions to circumvent this problem, including lipid-coated lipid-coated microrods\cite{Cheung2018}, 3D-scaffolds via either
microrods\cite{Cheung2018}, 3D-scaffolds via either Matrigel\cite{Rio2018} or Matrigel\cite{Rio2018} or 3d-printed lattices\cite{Delalat2017}, ellipsoid
3d-printed lattices\cite{Delalat2017}, ellipsoid beads\cite{meyer15_immun}, and beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms}
\gls{mab}-conjugated \gls{pdms} beads\cite{Lambert2017} that respectively beads\cite{Lambert2017} that respectively recapitulate the cellular membrane,
recapitulate the cellular membrane, large interfacial contact area, large interfacial contact area, 3D-structure, or soft surfaces T cells normally
3D-structure, or soft surfaces T cells normally experience \textit{in vivo}. experience \textit{in vivo}. None of these have been shown to expand high
None have been demonstrated to demonstrably expand high quality T cells as quality T cells as outlined in \cref{sec:background_quality}.
outlined in \cref{sec:background_quality}.
\subsection{Microcarriers in Bioprocessing} \subsection{Microcarriers in Bioprocessing}
@ -1046,56 +1043,54 @@ methods described in \cref{sec:background_activation}.
Microcarriers have historically been used to grow a number of adherent cell Microcarriers have historically been used to grow a number of adherent cell
types for a variety of applications. They were introduced in 1967 as a means to types for a variety of applications. They were introduced in 1967 as a means to
grow adherent cells `in suspension', effectively turning a 2D flask system into grow adherent cells ``in suspension,'' effectively turning a 2D flask into a 3D
a 3D culture system\cite{WEZEL1967}. Microcarriers are generally spherical and culture system\cite{WEZEL1967}. Microcarriers are generally spherical and are
are several hundred \si{\um} in diameter, which means they collectively have a several hundred \si{\um} in diameter, which means they collectively have a much
much higher surface area than a traditional flask when matched for volume. higher surface area than a traditional flask when matched for volume.
Consequently, this means that microcarrier-based cultures can operate with much Consequently, this means that microcarrier-based cultures can operate with much
lower footprints than flask-like systems. Microcarriers also allow cell culture lower footprints than flask-like systems. Microcarriers also allow cell cultures
to operate more like a traditional chemical engineering process, wherein a to operate more like traditional chemical engineering processes, wherein a
\gls{cstr} may be employed to enhance oxygen transfer, maintain pH, \gls{cstr} may be employed to enhance oxygen transfer, maintain pH, and
and continuously supply nutrients\cite{Derakhti2019}. continuously supply nutrients\cite{Derakhti2019}.
A variety of microcarriers have been designed, primarily differing in their A variety of microcarriers have been designed, primarily differing in their
choice of material and macroporous structure. Key concerns driving these choice of material and macroporous structure. Key concerns driving these choices
choiceshave been cell attachment at the beginning of culture and cell detachment have been cell attachment at the beginning of culture and cell detachment at the
at the harvesting step\cite{Derakhti2019}. Many microcarriers simply use harvesting step\cite{Derakhti2019}. Many microcarriers simply use polystyrene
polystyrene (the material used for tissue culture flasks and dishes in general) (the material used for tissue culture flasks and dishes in general) with no
with no modification (SoloHill Plastic, Nunc Biosilon), with a cationic surface modification (SoloHill Plastic, Nunc Biosilon), with a cationic surface charge
charge (SoloHill Hillex) or coated with an \gls{ecm} protein such as collagen (SoloHill Hillex) or coated with an \gls{ecm} protein such as collagen (SoloHill
(SoloHill Fact III). Other base materials have been used such as dextran (GE Fact III). Other base materials have been used such as dextran (GE Healthcare
Healthcare Cytodex), cellulose (GE Healthcare Cytopore), and glass (\sigald{} Cytodex), cellulose (GE Healthcare Cytopore), and glass (\sigald{} G2767), all
G2767), all with none or similar surface modifications. Additionally, some with similar surface modifications (if any). Additionally, some microcarriers
microcarriers such as \gls{cus} and \gls{cug} are composed entirely out of such as \gls{cus} and \gls{cug} are composed entirely out of protein (in these
protein (in these cases, porcine collagen) which also allows the microcarriers cases, porcine collagen) which also allows the microcarriers to be enzymatically
to be enzymatically degraded. In the case of non-protein materials, cells may degraded. In the case of non-protein materials, cells may still be detached
still be detached using enzymes but these require similar methods to those using enzymes but these require similar methods to those currently used in
currently used in flasks such as trypsin which target the cellular \gls{ecm} flasks such as trypsin which target the cellular \gls{ecm} directly. Since
directly. Since trypsin and related enzymes tends to be harsh on cells, an trypsin and related enzymes tends to be harsh on cells, an advantage of using
advantage of using entirely protein-based microcarriers is that they can be entirely protein-based microcarriers is that they can be degraded using a much
degraded using a much safer enzyme such as collagenase, at the cost of being safer enzyme such as collagenase, at the cost of being more expensive and also
more expensive and also being harder to make being harder to make \gls{gmp}-compliant\cite{Derakhti2019}. Going one step
\gls{gmp}-compliant\cite{Derakhti2019}. Going one step further, some further, some microcarriers are composed of a naturally degrading scaffold such
microcarriers are composed of a naturally degrading scaffold such as alginate, as alginate, which do not need an enzyme for degradation. Finally, microcarriers
which do not need an enzyme for degradation. Finally, microcarriers can differ can differ in their overall structure. \gls{cug} and \gls{cus} microcarriers as
in their overall structure. \gls{cug} and \gls{cus} microcarriers as well as the well as the Cytopore microcarriers are macroporous, meaning they have a porous
Cytopore microcarriers are macroporous, meaning they have a porous network in network in which cells can attach throughout their interior. This drastically
which cells can attach throughout their interior. This drastically increases the increases the effective surface area and consequently the number of cells which
effective surface area and consequently the number of cells which may be grown may be grown per unit volume. Other microcarriers are microporous (eg only
per unit volume. Other microcarriers are microporous (eg only to small permeable to small molecules) or not porous at all; in either case, cells can
molecules) or not porous at all (eg polystyrene); in either case the cells can only grow on the outer surface.
only grow on the surface.
Microcarriers in general have seen the most use in growing \gls{cho} cells and Microcarriers have been mainly used for growing \gls{cho} cells and hybridomas
hybridomas in the case of protein manufacturing (eg \gls{igg} in the case of protein manufacturing (eg \gls{igg} production)\cite{Xiao1999,
production)\cite{Xiao1999, Kim2011} as well as \glspl{esc} and \glspl{msc} more Kim2011} as well as \glspl{esc} and \glspl{msc} more recently in the case of
recently in the case of cell manufacturing\cite{Heathman2015, Sart2011, cell manufacturing\cite{Heathman2015, Sart2011, Chen2013, Schop2010, Rafiq2016}.
Chen2013, Schop2010, Rafiq2016}. Interestingly, some groups have even explored Interestingly, some groups have even explored using biodegradable microcarriers
using biodegradable microcarriers \invivo{} as a delivery vehicle for stem cell \invivo{} as a delivery vehicle for stem cell therapies in the context of
therapies in the context of regenerative medicine\cite{Zhang2016, Saltz2016, regenerative medicine\cite{Zhang2016, Saltz2016, Park2013, Malda2006}. However,
Park2013, Malda2006}. However, the characteristic shared by all the cell types all these cell types are adherent. In this work, we explore the use of
in this application is the fact that they are adherent. In this work, we explore microcarrier for T cells, which are naturally non-adherent.
the use of microcarrier for T cells, which are naturally non-adherent.
The microcarriers used in this work were \gls{cus} and \gls{cug} (mostly the The microcarriers used in this work were \gls{cus} and \gls{cug} (mostly the
former) which are both composed of cross-linked gelatin and have a macroporous former) which are both composed of cross-linked gelatin and have a macroporous
@ -1119,7 +1114,7 @@ T cells naturally expand in the lymph nodes which have an \gls{ecm} composed of
collagen\cite{Dustin2001, Ebnet1996, Ohtani2008}. Despite this, T cells don't collagen\cite{Dustin2001, Ebnet1996, Ohtani2008}. Despite this, T cells don't
interact with collagen fibers in the lymph node as the collagen fibers are interact with collagen fibers in the lymph node as the collagen fibers are
sheathed with stromal fibroblasts\cite{Dustin2001, Ebnet1996}. However, the sheathed with stromal fibroblasts\cite{Dustin2001, Ebnet1996}. However, the
\gls{ecm} of peripheral tissues is dense with exposed collagen fibers are \gls{ecm} of peripheral tissues is dense where exposed collagen fibers are
available to interact with T cells. Furthermore, T cells have been shown available to interact with T cells. Furthermore, T cells have been shown
\invitro{} to crawl along collagen fibers in the presence of \glspl{apc}, \invitro{} to crawl along collagen fibers in the presence of \glspl{apc},
facilitating short encounters with \glspl{apc}\cite{Gunzer2000}. While this may facilitating short encounters with \glspl{apc}\cite{Gunzer2000}. While this may
@ -1129,16 +1124,15 @@ not be ideal \invivo{} due to the lack of cumulative signal received by
The major surface receptors for collagen are \gls{a2b1} and The major surface receptors for collagen are \gls{a2b1} and
\gls{a2b2}\cite{Dustin2001, Hemler1990}. These receptors are not expressed on \gls{a2b2}\cite{Dustin2001, Hemler1990}. These receptors are not expressed on
naive T cells and thus presence and stimulation of collagen alone is not naive \gls{tn} cells and thus presence and stimulation of collagen alone is not
sufficient to cause activation or expansion of T cells\cite{Hemler1990}. These sufficient for activation or expansion\cite{Hemler1990}; however, they have been
receptors have been shown to lead to a number of functions that may be useful in shown to possess many functions that may be useful for T cell expansion. First,
the context of T cell expansion. First, they have been shown to act in a they can act in a costimulatory manner which leads to increased
costimulatory manner which leads to increased proliferation\cite{Rao2000}. proliferation\cite{Rao2000}. Furthermore, \gls{a2b1} and \gls{a2b2} seem to
Furthermore, \gls{a2b1} and \gls{a2b2} have been shown to protect Jurkat cells protect Jurkat cells against Fas-mediated apoptosis in the presence of collagen
against Fas-mediated apoptosis in the presence of collagen I\cite{Aoudjit2000, I\cite{Aoudjit2000, Gendron2003}. Finally, these receptors can increase
Gendron2003}. Finally, these receptors have been shown to increase \gls{ifng} \gls{ifng} production \invitro{} when T cells derived from human \glspl{pbmc}
production \invitro{} when T cells derived from human \glspl{pbmc} are are stimulated in the presence of collagen I\cite{Boisvert2007}.
stimulated in the presence of collagen I\cite{Boisvert2007}.
\subsection{The Role of IL15 in Memory T Cell Proliferation} \subsection{The Role of IL15 in Memory T Cell Proliferation}
@ -1148,8 +1142,8 @@ further exploration in \cref{aim2b}.
Functionally, mice lacking the gene for either \il{15}\cite{Kennedy2000} or its Functionally, mice lacking the gene for either \il{15}\cite{Kennedy2000} or its
high affinity receptor \il{15R$\upalpha$}\cite{Lodolce1998} are generally high affinity receptor \il{15R$\upalpha$}\cite{Lodolce1998} are generally
healthy but show a deficit in memory CD8 T cells, thus underscoring its healthy but show a deficit in memory CD8 T cells, thus underscoring this
importance in manufacturing high-quality memory T cells for immunotherapies. T cytokine's importance in producing memory T cells for immunotherapies. T
cells themselves express \il{15} and all of its receptor cells themselves express \il{15} and all of its receptor
components\cite{MirandaCarus2005}. Additionally, blocking \il{15} itself or components\cite{MirandaCarus2005}. Additionally, blocking \il{15} itself or
\il{15R$\upalpha$} \invitro{} has been shown to inhibit homeostatic \il{15R$\upalpha$} \invitro{} has been shown to inhibit homeostatic
@ -1158,20 +1152,20 @@ proliferation of resting human T cells\cite{MirandaCarus2005}.
% ACRO fix the il2R and IL15R stuff % ACRO fix the il2R and IL15R stuff
\il{15} has been puzzling historically because it shares almost the same pathway \il{15} has been puzzling historically because it shares almost the same pathway
as \il{2} yet has different functions\cite{Stonier2010, Osinalde2014, Giri1994, as \il{2} yet has different functions\cite{Stonier2010, Osinalde2014, Giri1994,
Giri1995}. In particular, both cytokines share the common $\upgamma$ subchain Giri1995}. In particular, both cytokines bond with heterotrimeric receptors
(CD132) as well as the \il{2} $\upbeta$ receptor (CD122). The main difference in which share the common $\upgamma$ subchain (CD132) as well as the \il{2}
the heterodimeric receptors for \il{2} and \il{15} is the \il{2} $\upalpha$ $\upbeta$ receptor (CD122). The difference is the third subchain which is either
receptor (CD25) and the \il{15} $\upalpha$ chain respectively, both of which the \il{2} $\upalpha$ receptor (CD25) or the \il{15} $\upalpha$ chain
have high affinity for their respective ligands. The \il{2R$\upalpha$} chain respectively, both of which have high affinity for their respective ligands. The
itself does not have any signaling capacity, and therefore all the signaling \il{2R$\upalpha$} chain itself does not have any signaling capacity, and
resulting from \il{2} is mediated thought the $\upbeta$ and $\upgamma$ chains, therefore all the signaling resulting from \il{2} is mediated thought the
namely via JAK1 and JAK3 leading to STAT5 activation consequently T cell $\upbeta$ and $\upgamma$ chains (namely via JAK1 and JAK3, which leads to STAT5
activation. \il{15R$\upalpha$} itself has some innate signaling capacity, but activation, which leads to T cell activation). \il{15R$\upalpha$} itself has
this is poorly characterized in lymphocytes\cite{Stonier2010}. Thus there is a some innate signaling capacity, but this is poorly characterized in
significant overlap between the functions of \il{2} and \il{15} due to their lymphocytes\cite{Stonier2010}. Thus there is a significant overlap between the
receptors sharing the $\upbeta$ and $\upgamma$ chains in their heterodimeric functions of \il{2} and \il{15} due to their receptors sharing the $\upbeta$ and
receptors, and perhaps the main driver of their differential functions it the $\upgamma$ chains, and perhaps the main driver of their differential functions
half life of each respective receptor\cite{Osinalde2014}. it the half life of each respective receptor\cite{Osinalde2014}.
Where \il{15} is unique is that many (or possibly most) of its functions derive Where \il{15} is unique is that many (or possibly most) of its functions derive
from being membrane-bound to its receptor\cite{Stonier2010}. Particularly, from being membrane-bound to its receptor\cite{Stonier2010}. Particularly,
@ -1186,18 +1180,18 @@ mechanism is that cells expression \il{15R$\upalpha$} either need to express
proximity require the $\upbeta$ and $\upgamma$ chains to receive the signal. In proximity require the $\upbeta$ and $\upgamma$ chains to receive the signal. In
addition to \textit{trans} presentation, \il{15} may also work in a \textit{cis} addition to \textit{trans} presentation, \il{15} may also work in a \textit{cis}
manner, where \il{15R$\upalpha$}/\il{15} complexes may bind to the $\upbeta$ and manner, where \il{15R$\upalpha$}/\il{15} complexes may bind to the $\upbeta$ and
$\upgamma$ chains on the same cell, assuming all receptors are expressed and $\upgamma$ chains on the same cell, assuming each subchain is expressed and
soluble \il{15} is available\cite{Olsen2007}. Finally, \il{15R$\upalpha$} itself can exist in soluble \il{15} is available\cite{Olsen2007}. Finally, \il{15R$\upalpha$} itself
a soluble form, which can bind to \il{15} and signal to cells which are not can exist in soluble form, which can bind to \il{15} and signal to cells which
adjacent to the source independent of the \textit{cis/trans} mechanisms already are not adjacent to the source independent of the \textit{cis/trans} mechanisms
described\cite{Budagian2004}. already described\cite{Budagian2004}.
\subsection{Overview of Design of Experiments}\label{sec:background_doe} \subsection{Overview of Design of Experiments}\label{sec:background_doe}
The \gls{dms} system described in this dissertation has a number of parameters The \gls{dms} system described in this dissertation has many parameters that can
that can be optimized and controlled (eg \glspl{cpp}). A \gls{doe} is an ideal be optimized and controlled (eg \glspl{cpp}). A \gls{doe} is an ideal framework
framework to test multiple parameters simultaneously and determine which are to test multiple parameters simultaneously and determine which are relevant
relevant \glspl{cpp}. \glspl{cpp}.
The goal of \gls{doe} is to answer a data-driven question with the least number The goal of \gls{doe} is to answer a data-driven question with the least number
of resources\cite{Wu2009}. It was developed in many non-biological industries of resources\cite{Wu2009}. It was developed in many non-biological industries
@ -1206,30 +1200,33 @@ industries where engineers needed to minimize downtime and resource consumption
on full-scale production lines. on full-scale production lines.
At its core, a \gls{doe} is simply a matrix of conditions to test where each row At its core, a \gls{doe} is simply a matrix of conditions to test where each row
(usually called a `run') corresponds to one experimental unit for which the (usually called a ``run,'' which is the term used throughout this work)
conditions are applied, and each column represents a parameter of concern to be corresponds to one experimental unit for which the conditions are applied, and
tested. The values in each cell represent the level at which each parameter is each column represents a parameter of concern to be tested. The values in each
to be tested. When the experiment is performed using this matrix of conditions, cell represent the level of each parameter. When the experiment is performed
the results are be summarized into one or more `responses' that correspond to using this matrix of conditions, the results are be summarized into one or more
each run. These responses are then be modeled (usually using linear regression) ``responses'' that correspond to each run. These responses are then be modeled
to determine the statistic relationship (also called an `effect') between each (usually using linear regression) to determine the statistical relationship
parameter and the response(s). (also called an ``effect'') between each parameter and the response(s).
Collectively, the space spanned by all parameters at their feasible ranges is Collectively, the space spanned by all parameters at their feasible ranges is
commonly referred to as the `design space', and generally the goal of a commonly referred to as the ``design space'', and generally the goal of a
\gls{doe} is to explore this design space using using the least number of runs \gls{doe} is to explore this design space using using the least number of runs
possible. While there are many types of \glspl{doe} depending on the nature possible. While there are many types of \glspl{doe} depending on the nature
of the parameters and the goal of the experimenter, they all share common of the parameters and the goal of the experimenter, they all share common
principles: principles:
\begin{description} \begin{description}
\item [randomization --] The order in which the runs are performed should \item [randomization --] The order in which the runs are performed should be
ideally be as random as possible. This is to mitigate against any confounding randomized. This is to guarantee that the tested parameters are independent of
factors that may be present which depend on the order or position of the runs. any unobserved influences to the response, and thus allows the causal effect
For an example in context, the evaporation rate of media in a tissue culture of each parameter to be isolated completely\footnote{this is why \glspl{doe}
plate will be much faster at the perimeter of the plate vs the center. While are sometimes called ``black box models;'' one can can safely say ``this
randomization does not eliminate this error, it will ensure the error is parameter causes that'' without paying attention to the causal structure of
`spread' evenly across all runs in an unbiased manner. the experiment}. For an example in context, the evaporation rate of media in
a tissue culture plate will be much faster at the perimeter of the plate vs
the center. While randomization does not eliminate this error, it will ensure
the error is ``spread'' across all runs in an unbiased manner.
\item [replication --] Since the analysis of a \gls{doe} is inherently \item [replication --] Since the analysis of a \gls{doe} is inherently
statistical, replicates should be used to ensure that the underlying statistical, replicates should be used to ensure that the underlying
distribution of errors can be estimated. While this is not strictly necessary distribution of errors can be estimated. While this is not strictly necessary
@ -1237,7 +1234,7 @@ principles:
strong assumptions about the model structure (particularly in the case of strong assumptions about the model structure (particularly in the case of
high-complexity models which could easily fit the data perfectly) and also high-complexity models which could easily fit the data perfectly) and also
precludes the use of statistical tests such as the lack-of-fit test which can precludes the use of statistical tests such as the lack-of-fit test which can
be useful in rejecting or accepting a particular analysis. Note that the be useful in rejecting or accepting a particular model. Note that the
subject of replication is within but not the same as power analysis, which subject of replication is within but not the same as power analysis, which
concerns the number of runs required to estimate a certain effect size. concerns the number of runs required to estimate a certain effect size.
\item [orthogonality --] Orthogonality refers to the independence of each \item [orthogonality --] Orthogonality refers to the independence of each
@ -1249,14 +1246,15 @@ principles:
experiments with many categorical variables) strategies exist to maximize experiments with many categorical variables) strategies exist to maximize
orthogonality. orthogonality.
\item [blocking --] In the case where the experiment must be non-randomly spread \item [blocking --] In the case where the experiment must be non-randomly spread
over multiple groups, runs are assigned to `blocks' which are not necessarily over multiple groups, runs are assigned to ``blocks'' which are not
relevant to the goals of the experiment but nonetheless could affect the necessarily relevant to the goals of the experiment but nonetheless could
response. A key assumption that is (usually) made in the case of blocking is affect the response. A key assumption that is (usually) made in the case of
that there is no interaction between the blocking variable and any of the blocking is that there is no interaction between the blocking variable and any
experimental parameters. For example, in T cell expansion, if media lot were a of the experimental parameters. For example, in T cell expansion, if media lot
blocking variable and expansion method were a parameter, we would by default were a blocking variable and expansion method were a parameter, we would by
assume that the effect of the expansion method does not depend on the media default assume that the effect of the expansion method does not depend on the
lot (even if the media lot itself might change the mean of the response). media lot (even if the media lot itself might change the mean of the
response).
\end{description} \end{description}
\Glspl{doe} served three purposes in this dissertation. First, we used them as \Glspl{doe} served three purposes in this dissertation. First, we used them as
@ -1279,28 +1277,26 @@ investigated. However, it could be the case that one already has data on many of
the factors of concern. If one only cares about main effects, performing a the factors of concern. If one only cares about main effects, performing a
\gls{doe} (particularly a lower-powered screening experiment such as a \gls{doe} (particularly a lower-powered screening experiment such as a
resolution III design) with these factors and a few others may not be resolution III design) with these factors and a few others may not be
productive, and one is better off performed a few extra pilot experiments before productive, and one is better off performing a few extra pilot experiments
doing a more complex design such as a central composite if desired. Furthermore, before doing a more complex design such as a central composite if desired.
it should be noted that while the goal of a \gls{doe} is to minimize resources, Furthermore, it should be noted that while the goal of a \gls{doe} is to
the size necessary to justify a \gls{doe} may not be worth the experimental minimize resources, the size necessary to justify a \gls{doe} may not be worth
return. For biological work (or any domain with little automation), performing a the experimental return. For biological work (or any domain with little
randomized experiment with 20 to 30 runs is not trivial from a logistical automation), performing a randomized experiment with 20 to 30 runs is not
perspective, especially when considering the number of manual manipulations and trivial from a logistical perspective, especially when considering the number of
the chance of human error. manual manipulations and the chance of human error.
Despite these caveats, many of the principles used for a \gls{doe} are important Despite these caveats, many of the principles used for a \gls{doe} are important
in general for experimentation. The most obvious is randomization, which is in general for experimentation. The most obvious is randomization, which is
often not employed (and also not explicitly mentioned in papers) even though it often not employed (and also not explicitly mentioned in papers). Assuming the
is empirically obvious that well plates have different evaporation rates experiment is manual, the largest reason to avoid randomization is that the
depending on well position. Assuming the experiment is manual, the largest experimentalist has no pattern to follow when administering treatment (such as
reason to avoid randomization is that the experimentalist has no pattern to ``add X to row 1 in well plate''), thus cognitive burden and the likelihood of
follow when administering treatment (such as ``add X to row 1 in well plate''), mistakes increases. While \glspl{doe} are usually bigger with more parameters,
thus cognitive burden and the likelihood of mistakes increases. While the one-factor-at-a-time experiment usually performed in biological disciplines
\glspl{doe} are usually bigger with more parameters, the one-factor-at-a-time is much smaller and only has a few parameters, thus these concerns are minimal.
experiment usually performed in biological disciplines is much smaller and only There is no reason to avoid randomization in these cases, as the added cognitive
has a few parameters, thus these concerns are minimal. There is no reason to cost is far offset by the guarantee of eliminated bias due to run position.
avoid randomization in these cases, as the added cognitive cost is far offset by
the guarantee of eliminated bias due to run position.
\subsection{Identification and Standardization of CPPs and \subsection{Identification and Standardization of CPPs and
CQAs}\label{sec:background_cqa} CQAs}\label{sec:background_cqa}
@ -1315,15 +1311,15 @@ secrete numerous cytokines and metabolites in the media, which may reflect the
internal state accurately and thus serve as a potential set of \glspl{cqa}. internal state accurately and thus serve as a potential set of \glspl{cqa}.
The complexity of these pathways dictates that we take a big-data approach to The complexity of these pathways dictates that we take a big-data approach to
this problem. To this end, there are several pertinent multi-omic (or simply this problem. To this end, there are several multi-omic (or simply ``omic'')
`omic') techniques that can be used to collect such datasets, which can then be techniques that can be used to collect such datasets, which can then be fit to
mined, modeled, and correlated to relevent responses (such as an endpoint relevent responses (such as an endpoint quantification of memory T cells) to
quantification of memory T cells) to identify pertinent \glspl{cqa}. identify pertinent \glspl{cqa}.
An overview of the techniques used in this work are: An overview of the techniques used in this work are:
\begin{description} \begin{description}
\item[Luminex --] This is a multiplexed bead-based assay similar to \gls{elisa} that can measure \item[luminex --] This is a multiplexed bead-based assay similar to \gls{elisa} that can measure
many bulk (not single cell) cytokine concentrations simultaneously many bulk (not single cell) cytokine concentrations simultaneously
in a media sample. This is a destructive assay but does not require cells to in a media sample. This is a destructive assay but does not require cells to
obtain a measurement. obtain a measurement.
@ -1333,7 +1329,7 @@ An overview of the techniques used in this work are:
oxidation\cite{Buck2016, van_der_Windt_2012}. \gls{nmr} is a technique that oxidation\cite{Buck2016, van_der_Windt_2012}. \gls{nmr} is a technique that
can non-destructively quantify small molecules in a media sample, and thus is can non-destructively quantify small molecules in a media sample, and thus is
an attractive method that could be used for inline, real-time monitoring. an attractive method that could be used for inline, real-time monitoring.
\item[Flow and Mass Cytometry --] Flow cytometry using fluorophores has been \item[flow and mass cytometry --] Flow cytometry using fluorophores has been
used extensively for immune cell analysis, but has a practical limit of used extensively for immune cell analysis, but has a practical limit of
approximately 18 colors\cite{Spitzer2016}. Mass cytometry is analogous to approximately 18 colors\cite{Spitzer2016}. Mass cytometry is analogous to
traditional flow cytometry except that it uses heavy-metal \gls{mab} traditional flow cytometry except that it uses heavy-metal \gls{mab}
@ -1359,11 +1355,11 @@ interesting cell types and the markers that define them.
Ultimately, identifying \glspl{cqa} will likely be an iterative process, wherein Ultimately, identifying \glspl{cqa} will likely be an iterative process, wherein
putative \glspl{cqa} will be identified, the corresponding \glspl{cpp} will be putative \glspl{cqa} will be identified, the corresponding \glspl{cpp} will be
set and optimized to maximize products with these \glspl{cpp}, and then set to maximize high-quality products, and then additional data will be
additional data will be collected in the clinic as the product is tested on collected in the clinic as the product is tested on various patients with
various patients with different indications. Additional \glspl{cqa} may be different indications. Additional \glspl{cqa} may be identified which better
identified which better predict specific clinical outcomes, which can be fed predict specific clinical outcomes, which can be fed back into the process model
back into the process model and optimized again. and optimized again.
\section{Innovation} \section{Innovation}
@ -1373,7 +1369,7 @@ novel considering the state-of-the-art technology for T cell manufacturing:
\begin{itemize} \begin{itemize}
\item \Glspl{dms} offers a compelling alternative to state-of-the-art magnetic \item \Glspl{dms} offers a compelling alternative to state-of-the-art magnetic
bead technologies (e.g. DynaBeads, MACS-Beads), which is noteworthy because bead technologies (e.g. DynaBeads, MACS-Beads), which is noteworthy because
the licenses for these techniques is controlled by only a few companies the licenses for these techniques are controlled by only a few companies
(Invitrogen and Miltenyi respectively). Because of this, bead-based expansion (Invitrogen and Miltenyi respectively). Because of this, bead-based expansion
is more expensive to implement and therefore hinders companies from entering is more expensive to implement and therefore hinders companies from entering
the rapidly growing T cell manufacturing arena. Providing an alternative will the rapidly growing T cell manufacturing arena. Providing an alternative will
@ -1381,7 +1377,7 @@ novel considering the state-of-the-art technology for T cell manufacturing:
lower costs, and higher innovation in the T cell manufacturing space. lower costs, and higher innovation in the T cell manufacturing space.
\item This is the first technology for T cell immunotherapies that selectively \item This is the first technology for T cell immunotherapies that selectively
expands memory T cell populations with greater efficiency relative to expands memory T cell populations with greater efficiency relative to
bead-based expansion Others have demonstrated methods that can achieve greater bead-based expansion. Others have demonstrated methods that can achieve greater
expansion of T cells, but not necessarily specific populations that are known expansion of T cells, but not necessarily specific populations that are known
to be potent. to be potent.
\item We used \glspl{doe} to discover and validate novel \glspl{cpp}, which is a \item We used \glspl{doe} to discover and validate novel \glspl{cpp}, which is a
@ -1389,9 +1385,9 @@ novel considering the state-of-the-art technology for T cell manufacturing:
usage in the development of cell therapies where research often employs a usage in the development of cell therapies where research often employs a
one-factor-at-a-time approach. We believe this method is highly relevant to one-factor-at-a-time approach. We believe this method is highly relevant to
the development of cell therapies, not only for process optimization but also the development of cell therapies, not only for process optimization but also
hypotheses generation. Furthermore, it is a perfectly natural strategy to use hypotheses generation. Furthermore, it is a natural strategy to use even at
even at small scale, where the cost of reagents, cells, and materials often small scale, where the cost of reagents, cells, and materials often precludes
precludes large sample sizes. large sample sizes.
\item The \gls{dms} system is be compatible with static bioreactors such as the \item The \gls{dms} system is be compatible with static bioreactors such as the
G-Rex which has been adopted throughout the cell therapy industry. Thus this G-Rex which has been adopted throughout the cell therapy industry. Thus this
technology can be easily incorporated into existing cell therapy process that technology can be easily incorporated into existing cell therapy process that