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\usepackage[version=4]{mhchem}
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\usepackage{pgfgantt}
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\usepackage{setspace}
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\doublespacing{}
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\titleformat{\chapter}[block]{\filcenter\bfseries\large}
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{\MakeUppercase{\chaptertitlename} \thechapter: }{0pt}{\uppercase}
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% \titleformat{\chapter}[block]{\filcenter\bfseries\large}{}{0pt}{\uppercase}
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\titleformat{\subsubsection}[runin]{\bfseries\itshape\/}{}{0pt}{\titlecap}
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\DeclareSIUnit\activityunit{U}
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\DeclareSIUnit\carrier{carriers}
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\DeclareSIUnit\cell{cells}
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\DeclareSIUnit\ab{mAbs}
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% add acronyms here
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\renewcommand{\glossarysection}[2][]{} % remove glossary title
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\makeglossaries
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\newacronym{act}{ACT}{adoptive cell therapies}
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\newacronym{car}{CAR}{chimeric antigen receptor}
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\newacronym[longplural={monoclonal antibodies}]{mab}{mAb}{monoclonal antibody}
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\newacronym{ecm}{ECM}{extracellular matrix}
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\newacronym{cqa}{CQA}{critical quality attribute}
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\newacronym{cpp}{CPP}{critical process parameter}
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\newacronym{dms}{DMS}{degradable microscaffold}
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\newacronym{doe}{DOE}{design of experiments}
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\newacronym{gmp}{GMP}{Good Manufacturing Practices}
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\newacronym{cho}{CHO}{Chinese hamster ovary}
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\newacronym{all}{ALL}{acute lymphoblastic leukemia}
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\newacronym{pdms}{PDMS}{polydimethylsiloxane}
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\newacronym{dc}{DC}{dendritic cell}
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\newacronym{il2}{IL2}{interleukin 2}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% my commands
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\newcommand{\mytitle}{
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\Large{
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\textbf{
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Optimizing T Cell Manufacturing and Quality Using Functionalized
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Degradable Microscaffolds
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}
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}
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}
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\newcommand{\mycommitteemember}[3]{
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\begin{flushleft}
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\noindent
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#1 \\
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#2 \\
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\textit{#3}
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\end{flushleft}
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}
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\newcommand{\invivo}{\textit{in vivo}}
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\newcommand{\invitro}{\textit{in vitro}}
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\newcommand{\exvivo}{\textit{ex vivo}}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% my environments
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\newenvironment{mytitlepage}{
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}
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{
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\end{singlespace}
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}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% document
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\begin{document}
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\begin{titlepage}
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\begin{mytitlepage}
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\mytitle{}
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\vfill
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\Large{
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A Dissertation \\
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Presented to \\
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The Academic Faculty \\
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\vspace{1.5em}
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by
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\vspace{1.5em}
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Nathan John Dwarshuis, B.S. \\
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\vfill
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In Partial Fulfillment \\
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of the Requirements for the Degree \\
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Doctor of Philosophy in Biomedical Engineering in the \\
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Wallace H. Coulter Department of Biomedical Engineering
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\vfill
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Georgia Institute of Technology and Emory University \\
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August 2021
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\vfill
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COPYRIGHT \copyright{} BY NATHAN J. DWARSHUIS
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}
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\end{mytitlepage}
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\end{titlepage}
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\onecolumn \pagenumbering{roman}
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\clearpage
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\begin{mytitlepage}
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\mytitle{}
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\end{mytitlepage}
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\vfill
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\large{
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\noindent
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Committee Members
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\begin{multicols}{2}
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\begin{singlespace}
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\mycommitteemember{Dr.\ Krishnendu\ Roy\ (Advisor)}
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{Department of Biomedical Engineering}
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{Georgia Institute of Technology and Emory University}
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\vspace{1.5em}
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\mycommitteemember{Dr.\ Madhav\ Dhodapkar}
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{Department of Hematology and Medical Oncology}
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{Emory University}
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\vspace{1.5em}
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\mycommitteemember{Dr.\ Melissa\ Kemp}
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{Department of Biomedical Engineering}
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{Georgia Institute of Technology and Emory University}
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\columnbreak{}
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\null{}
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\vfill
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\mycommitteemember{Dr.\ Wilbur\ Lam}
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{Department of Biomedical Engineering}
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{Georgia Institute of Technology and Emory University}
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\vspace{1.5em}
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\mycommitteemember{Dr.\ Sakis\ Mantalaris}
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{Department of Biomedical Engineering}
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{Georgia Institute of Technology and Emory University}
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\end{singlespace}
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\end{multicols}
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\vspace{1.5em}
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\hfill Date Approved:
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}
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\clearpage
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\chapter*{acknowledgements}
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\addcontentsline{toc}{chapter}{acknowledgements}
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Thank you to Lex Fridman and Devin Townsend for being awesome and inspirational.
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\clearpage
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\chapter*{summary}
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\addcontentsline{toc}{chapter}{summary}
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\Gls{act} using \gls{car} T cells have shown promise in treating cancer, but
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manufacturing large numbers of high quality cells remains challenging. Currently
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approved T cell expansion technologies involve anti-CD3 and CD28 \glspl{mab},
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usually mounted on magnetic beads. This method fails to recapitulate many key
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signals found \invivo{} and is also heavily licensed by a few companies,
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limiting its long-term usefulness to manufactures and clinicians. Furthermore,
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we understand that highly potent T cells are generally less-differentiated
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subtypes such as central memory and stem memory T cells. Despite this
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understanding, little has been done to optimize T cell expansion for generating
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these subtypes, including measurement and feedback control strategies that are
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necessary for any modern manufacturing process.
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The goal of this thesis was to develop a microcarrier-based \gls{dms} T cell
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expansion system as well as determine biologically-meaningful \glspl{cqa} and
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\glspl{cpp} that could be used to optimize for highly-potent T cells. In Aim 1,
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we develop and characterized the \gls{dms} system, including quality control
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steps. We also demonstrate the feasiblity of expanding highly-potent memory and
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CD4+ T cells, and showing compatibility with existing \gls{car} transduction
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methods. In aim 2, we use \gls{doe} methodology to optimize the \gls{dms}
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platform, and develop a computational pipeline to identify and model the effect
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of measurable \glspl{cqa} and \glspl{cpp} on the final product. In aim 3, we
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demonstrate the effectiveness of the \gls{dms} platform \invivo{}. This
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thesis lays the groundwork for a novel T cell expansion method which can be used
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in a clinical setting, and also provides a path toward optimizing for product
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quality in an industrial setting.
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\clearpage
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\tableofcontents
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\clearpage
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\listoffigures
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\clearpage
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\listoftables
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\clearpage
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% \twocolumn
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\chapter*{acronyms}
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\addcontentsline{toc}{chapter}{acronyms}
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\printglossary[type=\acronymtype]
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\clearpage
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\clearpage
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\chapter{introduction}
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\section*{overview}
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T cell-based immunotherapies have received great interest from clinicians and
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industry due to their potential to treat, and often cure, cancer and other
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diseases\cite{Fesnak2016,Rosenberg2015}. In 2017, Novartis and Kite Pharma
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received FDA approval for \textit{Kymriah} and \textit{Yescarta} respectively,
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two genetically-modified \gls{car} T cell therapies against B cell malignancies.
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Despite these successes, \gls{car} T cell therapies are constrained by an
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expensive and difficult-to-scale manufacturing process with little control on
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cell quality and phenotype3,4. State-of-the-art T cell manufacturing techniques
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focus on anti-CD3 and anti-CD28 activation and expansion, typically presented on
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superparamagnetic, iron-based microbeads (Invitrogen Dynabead, Miltenyi MACS
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beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers
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(Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017, Bashour2015}.
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These strategies overlook many of the signaling components present in the
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secondary lymphoid organs where T cells expand \invivo{}. Typically, T cells are
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activated under close cell-cell contact, which allows for efficient
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autocrine/paracrine signaling via growth-stimulating cytokines such as
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\gls{il2}. Additionally, the lymphoid tissues are comprised of \gls{ecm}
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components such as collagen, which provide signals to upregulate proliferation,
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cytokine production, and pro-survival pathways\cite{Gendron2003, Ohtani2008,
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Boisvert2007, Ben-Horin2004}. We hypothesized that culture conditions that
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better mimic these \invivo{} expansion conditions of T cells, can significantly
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improve the quality and quantity of manufactured T cells and provide better
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control on the resulting T cell phenotype.
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% TODO mention the Cloudz stuff that's in my presentation
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A variety of solutions have been proposed to make the T cell expansion process
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more physiological. One strategy is to use modified feeder cell cultures to
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provide activation signals similar to those of \glspl{dc}\cite{Forget2014}.
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While this has the theoretical capacity to mimic many components of the lymph
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node, it is hard to reproduce on a large scale due to the complexity and
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inherent variability of using cell lines in a fully \gls{gmp}-compliant manner.
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Others have proposed biomaterials-based solutions to circumvent this problem,
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including lipid-coated microrods\cite{Cheung2018}, 3D-scaffolds via either
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Matrigel\cite{Rio2018} or 3d-printed lattices\cite{Delalat2017}, ellipsoid
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beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms}
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beads\cite{Lambert2017} that respectively recapitulate the cellular membrane,
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large interfacial contact area, 3D-structure, or soft surfaces T cells normally
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experience \invivo{}. While these have been shown to provide superior expansion
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compared to traditional microbeads, none of these methods has been able to show
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preferential expansion of functional naïve/memory and CD4 T cell populations.
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Generally, T cells with a lower differentiation state such as naïve and memory
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cells have been shown to provide superior anti-tumor potency, presumably due to
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their higher potential to replicate, migrate, and engraft, leading to a
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long-term, durable response\cite{Xu2014, Fraietta2018, Gattinoni2011,
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Gattinoni2012}. Likewise, CD4 T cells are similarly important to anti-tumor
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potency due to their cytokine release properties and ability to resist
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exhaustion\cite{Wang2018, Yang2017}. Therefore, methods to increase naïve/memory
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and CD4 T cells in the final product are needed, a critical consideration being
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ease of translation to industry and ability to interface with scalable systems
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such as bioreactors.
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% TODO probably need to address some of the modeling stuff here as well
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This thesis describes a novel degradable microscaffold-based method derived from
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porous microcarriers functionalized with anti-CD3 and anti-CD28 \glspl{mab} for
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use in T cell expansion cultures. Microcarriers have historically been used
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throughout the bioprocess industry for adherent cultures such as stem cells and
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\gls{cho} cells, but not with suspension cells such as T
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cells\cite{Heathman2015, Sart2011}. The microcarriers chosen to make the DMSs in
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this study have a microporous structure that allows T cells to grow inside and
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along the surface, providing ample cell-cell contact for enhanced autocrine and
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paracrine signaling. Furthermore, the carriers are composed of gelatin, which is
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a collagen derivative and therefore has adhesion domains that are also present
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within the lymph nodes. Finally, the 3D surface of the carriers provides a
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larger contact area for T cells to interact with the \glspl{mab} relative to
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beads; this may better emulate the large contact surface area that occurs
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between T cells and \glspl{dc}. These microcarriers are readily available in
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over 30 countries and are used in an FDA fast-track-approved combination retinal
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pigment epithelial cell product (Spheramine, Titan Pharmaceuticals) {\#}[Purcell
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documentation]. This regulatory history will aid in clinical translation. We
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show that compared to traditional microbeads, \gls{dms}-expanded T cells not
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only provide superior expansion, but consistently provide a higher frequency of
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naïve/memory and CD4 T cells (CCR7+CD62L+) across multiple donors. We also
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demonstrate functional cytotoxicity using a CD19 \gls{car} and a superior
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performance, even at a lower \gls{car} T cell dose, of \gls{dms}-expanded
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\gls{car}-T cells \invivo{} in a mouse xenograft model of human B cell \gls{all}.
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Our results indicate that \glspl{dms} provide a robust and scalable platform for
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manufacturing therapeutic T cells with higher naïve/memory phenotype and more
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balanced CD4+ T cell content.
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2021-07-22 11:30:00 -04:00
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\section*{hypothesis}
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The hypothesis of this dissertation was that using \glspl{dms} created from
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off-the-shelf microcarriers and coated with activating \glspl{mab} would lead to
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higher quantity and quality T cells as compared to state-of-the-art bead-based
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expansion. The objective of this dissertation was to develop this platform, test
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its effectiveness both \invivo{} and \invivo{}, and develop computational
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pipelines that could be used in a manufacturing environment.
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\section*{specific aims}
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The specific aims of this dissertation are outlined in
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\cref{fig:graphical_overview}.
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\begin{figure*}[ht!]
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\begingroup
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\includegraphics[width=\textwidth]{example-image-a}
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\endgroup
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\caption[Project Overview]{High-level workflow.}
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\label{fig:graphical_overview}
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\end{figure*}
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\subsection*{aim 1: develop and optimize a novel T cell expansion process that
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mimics key components of the lymph nodes}
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% TODO this might be easier to break apart in separate aims
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In this first aim, we demonstrated the process for manufacturing \glspl{dms},
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including quality control steps that are necessary for translation of this
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platform into a scalable manufacturing setting. We also demonstrate that the
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\gls{dms} platform leads to higher overall expansion of T cells and higher
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overall fractions of potent memory and CD4+ subtypes desired for T cell
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therapies. Finally, we demonstrate \invitro{} that the \gls{dms} platform can be
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used to generate functional \gls{car} T cells targeted toward CD19.
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\subsection*{aim 2: develop methods to control and predict T cell quality}
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For this second aim, we investigated methods to identify and control \glspl{cqa}
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and glspl{cpp} for manufacturing T cells using the \gls{dms} platform. This was
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accomplished through two sub-aims:
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\begin{itemize}
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\item[A --] Develop computational methods to control and predict T cell
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expansion and quality
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\item[B --] Perturb \gls{dms} expansion to identify additional mechanistic
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controls for expansion and quality
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\end{itemize}
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\subsection*{aim 3: confirm potency of T cells from novel T cell expansion
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process using \invivo{} xenograft mouse model}
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In this final aim, we demonstrate the effectiveness of \gls{dms}-expanded T
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cells compared to state-of-the-art beads using \invivo{} mouse models for
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\gls{all}.
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2021-07-22 11:30:00 -04:00
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\section*{outline}
|
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2021-07-22 11:30:00 -04:00
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\subsection*{Aim 1}
|
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Aim 1
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2021-07-22 11:30:00 -04:00
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\subsection*{Aim 2}
|
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Aim 2
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2021-07-22 11:30:00 -04:00
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\subsection*{Aim 3}
|
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Aim 3
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2021-07-22 11:30:00 -04:00
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\chapter{background and significance}
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\subsection*{background}
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\subsection*{current T cell manufacturing technologies}
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2021-07-09 12:39:33 -04:00
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bla bla
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2021-07-22 11:30:00 -04:00
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\section*{strategies to optimize cell manufacturing}
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2021-07-09 12:39:33 -04:00
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bla bla
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2021-07-22 11:30:00 -04:00
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\subsection*{strategies to characterize cell manufacturing}
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2021-07-09 12:39:33 -04:00
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bla bla
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2021-07-22 11:30:00 -04:00
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\section{Innovation}
|
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2021-07-22 11:30:00 -04:00
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\chapter{aim 1}
|
2021-07-09 12:39:33 -04:00
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2021-07-22 11:30:00 -04:00
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\section{introduction}
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\section{methods}
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\section{results}
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\section{discussion}
|
2021-07-09 12:39:33 -04:00
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2021-07-22 11:30:00 -04:00
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\chapter{Aim 2}
|
2021-07-09 12:39:33 -04:00
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2021-07-22 11:30:00 -04:00
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\section{introduction}
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\section{methods}
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\section{results}
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\section{discussion}
|
2021-07-09 12:39:33 -04:00
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2021-07-22 11:30:00 -04:00
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\chapter{Aim 3}
|
2021-07-09 12:39:33 -04:00
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2021-07-22 11:30:00 -04:00
|
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\section{introduction}
|
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|
\section{methods}
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\section{results}
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\section{discussion}
|
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2021-07-22 11:30:00 -04:00
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\chapter{conclusions and future work}
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\section{conclusions}
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\section{future work}
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2021-07-09 12:39:33 -04:00
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\onecolumn
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\clearpage
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% TODO some people put appendices here....not sure if I need to
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2021-07-22 11:30:00 -04:00
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\chapter{References}
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\renewcommand{\section}[2]{} % noop the original bib section header
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2021-07-22 13:14:35 -04:00
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\bibliography{references}
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\bibliographystyle{naturemag}
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\end{document}
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