ENH paraphrase background

tex/references.bib

@@ -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:

tex/thesis.tex

@@ -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
+cell quality and phenotype\cite{Roddie2019, Dwarshuis2017}. State-of-the-art T
-focus on \acd{3} and \acd{28} activation and expansion, typically
+cell manufacturing techniques focus on \acd{3} and \acd{28} activation and
-presented on superparamagnetic, iron-based microbeads (Invitrogen Dynabead,
+expansion, typically presented on superparamagnetic, iron-based microbeads
-Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers
+(Invitrogen Dynabead, Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or
-(Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017, Bashour2015}.
+in soluble tetramers (Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016,
-These strategies overlook many of the signaling components present in the
+  Piscopo2017, Bashour2015}. These strategies overlook many of the signaling
-secondary lymphoid organs where T cells expand \invivo{}. Typically, T cells are
+components present in the secondary lymphoid organs where T cells expand
-activated under close cell-cell contact, which allows for efficient
+\invivo{}. Typically, T cells are activated under close cell-cell contact, which
-autocrine/paracrine signaling via growth-stimulating cytokines such as
+allows for efficient autocrine/paracrine signaling via growth-stimulating
-\gls{il2}. Additionally, the lymphoid tissues are comprised of \gls{ecm}
+cytokines such as \gls{il2}. Additionally, the lymphoid tissues are comprised of
-components such as collagen, which provide signals to upregulate proliferation,
+\gls{ecm} components such as collagen and stromal cells, which provide signals
-cytokine production, and pro-survival pathways\cite{Gendron2003, Ohtani2008,
+to upregulate proliferation, cytokine production, and pro-survival
-  Boisvert2007, Ben-Horin2004}. We hypothesized that culture conditions that
+pathways\cite{Gendron2003, Ohtani2008, Boisvert2007, Ben-Horin2004}.
-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.
 
 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
+more physiological. Including feeder cell cultures\cite{Forget2014} and
-provide activation signals similar to those of \glspl{dc}\cite{Forget2014}.
+biomaterials-based methods such as lipid-coated microrods or 3D scaffold
-While this has the theoretical capacity to mimic many components of the lymph
+gels\cite{Cheung2018,Delalat2017,meyer15_immun,Lambert2017} that attempt to
-node, it is hard to reproduce on a large scale due to the complexity and
+recapitulate the cellular membrane, large interfacial contact area,
-inherent variability of using cell lines in a fully \gls{gmp}-compliant manner.
+3D-structure, or soft surfaces T cells normally experience \invivo{}. While
-Others have proposed biomaterials-based solutions to circumvent this problem,
+these have been shown to activation and expand T cells, they either are not
-including lipid-coated microrods\cite{Cheung2018}, 3D-scaffolds via either
+scalable (in the case of feeder cells) or still lack many of the signals and
-Matrigel\cite{Rio2018} or 3d-printed lattices\cite{Delalat2017}, ellipsoid
+cues T cells experience as the expand. Additionally, none have been shown to
-beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms}
+preferentially expand highly-potent T cell necessary for anti-cancer therapies.
-beads\cite{Lambert2017} that respectively recapitulate the cellular membrane,
+Such high potency cells including subtypes with low differentiation state such
-large interfacial contact area, 3D-structure, or soft surfaces T cells normally
+as \gls{tscm} and \gls{tcm} cells or CD4 cells, all of which have been shown to
-experience \invivo{}. While these have been shown to provide superior expansion
+be necessary for durable responses\cite{Xu2014, Fraietta2018, Gattinoni2011,
-compared to traditional microbeads, none of these methods has been able to show
+  Gattinoni2012,Wang2018, Yang2017}. Methods to increase memory and CD4 T cells
-preferential expansion of functional naïve/memory and CD4 T cell populations.
+in the final product are needed. Furthermore, \gls{qbd} principles such as
-Generally, T cells with a lower differentiation state such as naïve and memory
+discovering and validating novel \glspl{cqa} and \glspl{cpp} in the space of T
-cells have been shown to provide superior anti-tumor potency, presumably due to
+cell manufacturing are required to reproducibly manufacture these subtypes and
-their higher potential to replicate, migrate, and engraft, leading to a
+ensure low-cost and safe products with maximal effectiveness in the clinic
-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.
 
-% TODO probably need to address some of the modeling stuff here as well
+This dissertation describes a novel \acrlong{dms}-based method derived from
-
-This thesis describes a novel degradable microscaffold-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
+throughout the bioprocess industry for adherent cultures such as \gls{cho} cells
-\gls{cho} cells, but not with suspension cells such as T
+but not with suspension cells such as T cells\cite{Heathman2015, Sart2011}. The
-cells\cite{Heathman2015, Sart2011}. The microcarriers chosen to make the DMSs in
+microcarriers chosen to make the \gls{dms} in this work have a microporous
-this study have a microporous structure that allows T cells to grow inside and
+structure that allows T cells to grow inside and along the surface, providing
-along the surface, providing ample cell-cell contact for enhanced autocrine and
+ample cell-cell contact for enhanced autocrine and paracrine signaling.
-paracrine signaling. Furthermore, the carriers are composed of gelatin, which is
+Furthermore, the 3D surface of the carriers provides a larger contact area for T
-a collagen derivative and therefore has adhesion domains that are also present
+cells to interact with the \glspl{mab} relative to beads; this may better
-within the lymph nodes. Finally, the 3D surface of the carriers provides a
+emulate the large contact surface area that occurs between T cells and
-larger contact area for T cells to interact with the \glspl{mab} relative to
+\glspl{dc}.
-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.
 
 \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
+expansion. We also hypothesized that T cells have measurable biological
-its effectiveness both \invivo{} and \invivo{}, and develop computational
+signatures that are predictive of downstream outcomes and phenotypes. The
-pipelines that could be used in a manufacturing environment.
+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
+In \cref{background}, we provide additional background on the current state of T
-state of T cell manufacturing and how the work in this dissertation moves the
+cell manufacturing and how the work in this dissertation moves the field
-field forward. In Chapters~\ref{aim1},~\ref{aim2a},~\ref{aim2b}, and~\ref{aim3}
+forward. In \cref{aim1,aim2a,aim2b,aim3} we present the work pertaining to Aims
-we present the work pertaining to Aims 1, 2, and 3 respectively. Finally, we
+1, 2a, 2b, and 3 respectively. Finally, in \cref{conclusions} we present our
-present our final conclusions in Chapter~\ref{conclusions}.
+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
+Microcarriers in general have seen the most use in growing \gls{cho} cells and
-in the case of protein manufacturing (eg \gls{igg} production)\cite{Xiao1999,
+hybridomas in the case of protein manufacturing (eg \gls{igg}
-  Kim2011} as well as pluripotent stem cells and mesenchymal stromal cells more
+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