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@ -2596,6 +2596,57 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
publisher = {Springer Science and Business Media {LLC}},
}
@Article{Guerra2001,
author = {S. Del Guerra and C. Bracci and K. Nilsson and A. Belcourt and L. Kessler and R. Lupi and L. Marselli and P. De Vos and P. Marchetti},
journal = {Biotechnology and Bioengineering},
title = {Entrapment of dispersed pancreatic islet cells in {CultiSpher}-S macroporous gelatin microcarriers: Preparation, in vitro characterization, and microencapsulation},
year = {2001},
number = {6},
pages = {741--744},
volume = {75},
doi = {10.1002/bit.10053},
publisher = {Wiley},
}
@Article{Fernandes2007,
author = {A.M. Fernandes and T.G. Fernandes and M.M. Diogo and C. Lobato da Silva and D. Henrique and J.M.S. Cabral},
journal = {Journal of Biotechnology},
title = {Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system},
year = {2007},
month = {oct},
number = {2},
pages = {227--236},
volume = {132},
doi = {10.1016/j.jbiotec.2007.05.031},
publisher = {Elsevier {BV}},
}
@Article{Storm_2010,
author = {Michael P. Storm and Craig B. Orchard and Heather K. Bone and Julian B. Chaudhuri and Melanie J. Welham},
journal = {Biotechnology and Bioengineering},
title = {Three-dimensional culture systems for the expansion of pluripotent embryonic stem cells},
year = {2010},
month = {jun},
number = {4},
pages = {683--695},
volume = {107},
doi = {10.1002/bit.22850},
publisher = {Wiley},
}
@Article{Eibes2010,
author = {Gemma Eibes and Francisco dos Santos and Pedro Z. Andrade and Joana S. Boura and Manuel M.A. Abecasis and Cl{\'{a}}udia Lobato da Silva and Joaquim M.S. Cabral},
journal = {Journal of Biotechnology},
title = {Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system},
year = {2010},
month = {apr},
number = {4},
pages = {194--197},
volume = {146},
doi = {10.1016/j.jbiotec.2010.02.015},
publisher = {Elsevier {BV}},
}
@Comment{jabref-meta: databaseType:bibtex;}
@Comment{jabref-meta: grouping:

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tex/thesis.tex
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@ -1,4 +1,3 @@
% \documentclass[twocolumn]{article}
\documentclass{report}
\usepackage[section]{placeins}
\usepackage[top=1in,left=1.5in,right=1in,bottom=1in]{geometry}
@ -189,6 +188,8 @@
\newacronym{aws}{AWS}{amazon web services}
\newacronym{qpcr}{qPCR}{quantitative polymerase chain reaction}
\newacronym{cstr}{CSTR}{continuously stirred tank bioreactor}
\newacronym{esc}{ESC}{embryonic stem cell}
\newacronym{msc}{MSC}{mesenchymal stromal cells}
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% SI units for uber nerds
@ -455,9 +456,6 @@ quality in an industrial setting.
\section*{overview}
% TODO this is basically the same as the first part of the backgound, I guess I
% can just trim it down
T cell-based immunotherapies have received great interest from clinicians and
industry due to their potential to treat, and often cure, cancer and other
diseases\cite{Fesnak2016,Rosenberg2015}. In 2017, Novartis and Kite Pharma
@ -465,86 +463,63 @@ received FDA approval for \textit{Kymriah} and \textit{Yescarta} respectively,
two genetically-modified \gls{car} T cell therapies against B cell malignancies.
Despite these successes, \gls{car} T cell therapies are constrained by an
expensive and difficult-to-scale manufacturing process with little control on
cell quality and phenotype3,4. State-of-the-art T cell manufacturing techniques
focus on \acd{3} and \acd{28} activation and expansion, typically
presented on superparamagnetic, iron-based microbeads (Invitrogen Dynabead,
Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers
(Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017, Bashour2015}.
These strategies overlook many of the signaling components present in the
secondary lymphoid organs where T cells expand \invivo{}. Typically, T cells are
activated under close cell-cell contact, which allows for efficient
autocrine/paracrine signaling via growth-stimulating cytokines such as
\gls{il2}. Additionally, the lymphoid tissues are comprised of \gls{ecm}
components such as collagen, which provide signals to upregulate proliferation,
cytokine production, and pro-survival pathways\cite{Gendron2003, Ohtani2008,
Boisvert2007, Ben-Horin2004}. We hypothesized that culture conditions that
better mimic these \invivo{} expansion conditions of T cells, can significantly
improve the quality and quantity of manufactured T cells and provide better
control on the resulting T cell phenotype.
cell quality and phenotype\cite{Roddie2019, Dwarshuis2017}. State-of-the-art T
cell manufacturing techniques focus on \acd{3} and \acd{28} activation and
expansion, typically presented on superparamagnetic, iron-based microbeads
(Invitrogen Dynabead, Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or
in soluble tetramers (Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016,
Piscopo2017, Bashour2015}. These strategies overlook many of the signaling
components present in the secondary lymphoid organs where T cells expand
\invivo{}. Typically, T cells are activated under close cell-cell contact, which
allows for efficient autocrine/paracrine signaling via growth-stimulating
cytokines such as \gls{il2}. Additionally, the lymphoid tissues are comprised of
\gls{ecm} components such as collagen and stromal cells, which provide signals
to upregulate proliferation, cytokine production, and pro-survival
pathways\cite{Gendron2003, Ohtani2008, Boisvert2007, Ben-Horin2004}.
A variety of solutions have been proposed to make the T cell expansion process
more physiological. One strategy is to use modified feeder cell cultures to
provide activation signals similar to those of \glspl{dc}\cite{Forget2014}.
While this has the theoretical capacity to mimic many components of the lymph
node, it is hard to reproduce on a large scale due to the complexity and
inherent variability of using cell lines in a fully \gls{gmp}-compliant manner.
Others have proposed biomaterials-based solutions to circumvent this problem,
including lipid-coated microrods\cite{Cheung2018}, 3D-scaffolds via either
Matrigel\cite{Rio2018} or 3d-printed lattices\cite{Delalat2017}, ellipsoid
beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms}
beads\cite{Lambert2017} that respectively recapitulate the cellular membrane,
large interfacial contact area, 3D-structure, or soft surfaces T cells normally
experience \invivo{}. While these have been shown to provide superior expansion
compared to traditional microbeads, none of these methods has been able to show
preferential expansion of functional naïve/memory and CD4 T cell populations.
Generally, T cells with a lower differentiation state such as naïve and memory
cells have been shown to provide superior anti-tumor potency, presumably due to
their higher potential to replicate, migrate, and engraft, leading to a
long-term, durable response\cite{Xu2014, Fraietta2018, Gattinoni2011,
Gattinoni2012}. Likewise, CD4 T cells are similarly important to anti-tumor
potency due to their cytokine release properties and ability to resist
exhaustion\cite{Wang2018, Yang2017}. Therefore, methods to increase naïve/memory
and CD4 T cells in the final product are needed, a critical consideration being
ease of translation to industry and ability to interface with scalable systems
such as bioreactors.
more physiological. Including feeder cell cultures\cite{Forget2014} and
biomaterials-based methods such as lipid-coated microrods or 3D scaffold
gels\cite{Cheung2018,Delalat2017,meyer15_immun,Lambert2017} that attempt to
recapitulate the cellular membrane, large interfacial contact area,
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
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
preferentially expand highly-potent T cell necessary for anti-cancer therapies.
Such high potency cells including subtypes with low differentiation state such
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,
Gattinoni2012,Wang2018, Yang2017}. Methods to increase memory and CD4 T cells
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
cell manufacturing are required to reproducibly manufacture these subtypes and
ensure low-cost and safe products with maximal effectiveness in the clinic
% TODO probably need to address some of the modeling stuff here as well
This thesis describes a novel degradable microscaffold-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
use in T cell expansion cultures. Microcarriers have historically been used
throughout the bioprocess industry for adherent cultures such as stem cells and
\gls{cho} cells, but not with suspension cells such as T
cells\cite{Heathman2015, Sart2011}. The microcarriers chosen to make the DMSs in
this study have a microporous structure that allows T cells to grow inside and
along the surface, providing ample cell-cell contact for enhanced autocrine and
paracrine signaling. Furthermore, the carriers are composed of gelatin, which is
a collagen derivative and therefore has adhesion domains that are also present
within the lymph nodes. Finally, the 3D surface of the carriers provides a
larger contact area for T cells to interact with the \glspl{mab} relative to
beads; this may better emulate the large contact surface area that occurs
between T cells and \glspl{dc}. These microcarriers are readily available in
over 30 countries and are used in an FDA fast-track-approved combination retinal
pigment epithelial cell product (Spheramine, Titan
Pharmaceuticals)\cite{purcellmain}. This regulatory history will aid in clinical
translation. We show that compared to traditional microbeads, \gls{dms}-expanded
T cells not only provide superior expansion, but consistently provide a higher
frequency of naïve/memory and CD4 T cells (CCR7+CD62L+) across multiple donors.
We also demonstrate functional cytotoxicity using a CD19 \gls{car} and a
superior performance, even at a lower \gls{car} T cell dose, of
\gls{dms}-expanded \gls{car}-T cells \invivo{} in a mouse xenograft model of
human B cell \gls{all}. Our results indicate that \glspl{dms} provide a robust
and scalable platform for manufacturing therapeutic T cells with higher
naïve/memory phenotype and more balanced CD4+ T cell content.
throughout the bioprocess industry for adherent cultures such as \gls{cho} cells
but not with suspension cells such as T cells\cite{Heathman2015, Sart2011}. The
microcarriers chosen to make the \gls{dms} in this work have a microporous
structure that allows T cells to grow inside and along the surface, providing
ample cell-cell contact for enhanced autocrine and paracrine signaling.
Furthermore, the 3D surface of the carriers provides a larger contact area for T
cells to interact with the \glspl{mab} relative to beads; this may better
emulate the large contact surface area that occurs between T cells and
\glspl{dc}.
\section*{hypothesis}
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
higher quantity and quality T cells as compared to state-of-the-art bead-based
expansion. The objective of this dissertation was to develop this platform, test
its effectiveness both \invivo{} and \invivo{}, and develop computational
pipelines that could be used in a manufacturing environment.
expansion. We also hypothesized that T cells have measurable biological
signatures that are predictive of downstream outcomes and phenotypes. The
objective of this dissertation was to develop this platform, test its
effectiveness both \invitro{} and \invivo{}, and develop computational pipelines
to discover novel \glspl{cpp} and \glspl{cqa} that can be translated to a
manufacturing environment and a clinical trial setting.
\section*{specific aims}
@ -575,7 +550,7 @@ used to generate functional \gls{car} T cells targeted toward CD19.
\subsection*{aim 2: develop methods to control and predict T cell quality}
For this second aim, we investigated methods to identify and control \glspl{cqa}
and glspl{cpp} for manufacturing T cells using the \gls{dms} platform. This was
and \glspl{cpp} for manufacturing T cells using the \gls{dms} platform. This was
accomplished through two sub-aims:
\begin{itemize}
@ -594,11 +569,12 @@ cells compared to state-of-the-art beads using \invivo{} mouse models for
\section*{outline}
In Chapter~\ref{background}, we provide additional background on the current
state of T cell manufacturing and how the work in this dissertation moves the
field forward. In Chapters~\ref{aim1},~\ref{aim2a},~\ref{aim2b}, and~\ref{aim3}
we present the work pertaining to Aims 1, 2, and 3 respectively. Finally, we
present our final conclusions in Chapter~\ref{conclusions}.
In \cref{background}, we provide additional background on the current state of T
cell manufacturing and how the work in this dissertation moves the field
forward. In \cref{aim1,aim2a,aim2b,aim3} we present the work pertaining to Aims
1, 2a, 2b, and 3 respectively. Finally, in \cref{conclusions} we present our
conclusions as well as provide insights for how this work can be extended in the
future.
\chapter{background and significance}\label{background}
\section*{background}
@ -793,9 +769,9 @@ per unit volume. Other microcarriers are microporous (eg only to small
molecules) or not porous at all (eg polystyrene) in which case the cells can
only grow on the surface.
Microcarriers have seen the most use in growing \gls{cho} cells and hybridomas
in the case of protein manufacturing (eg \gls{igg} production)\cite{Xiao1999,
Kim2011} as well as pluripotent stem cells and mesenchymal stromal cells more
Microcarriers in general have seen the most use in growing \gls{cho} cells and
hybridomas in the case of protein manufacturing (eg \gls{igg}
production)\cite{Xiao1999, Kim2011} as well as \glspl{esc} and \glspl{msc} more
recently in the case of cell manufacturing\cite{Heathman2015, Sart2011,
Chen2013, Schop2010, Rafiq2016}. Interestingly, some groups have even explored
using biodegradable microcarriers \invivo{} as a delivery vehicle for stem cell
@ -804,6 +780,15 @@ 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
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
former) which are both composed of cross-linked gelatin and have a macroporous
morphology. These specific carriers have been used in the past for pancreatic
islet cells\cite{Guerra2001}, \glspl{esc}\cite{Fernandes2007, Storm_2010}, and
\glspl{msc}\cite{Eibes2010}. Furthermore, they are readily available in over 30
countries and are used in an FDA fast-track-approved combination retinal pigment
epithelial cell product (Spheramine, Titan Pharmaceuticals)\cite{purcellmain}.
This regulatory history will aid in clinical translation.
\subsection{methods to scale T cells}
In order to scale T cell therapies to meet clinical demands, automation and