\documentclass{report} \usepackage[top=1in,left=1.5in,right=1in,bottom=1in]{geometry} \usepackage[section]{placeins} \usepackage{siunitx} \usepackage{multicol} \setlength{\columnsep}{1cm} \usepackage[acronym,toc]{glossaries} \usepackage[T1]{fontenc} \usepackage{enumitem} \usepackage{titlesec} \usepackage{titlecaps} \usepackage{upgreek} \usepackage{graphicx} \usepackage{subcaption} \usepackage{nth} \usepackage[version=4]{mhchem} \usepackage{pgfgantt} \usepackage{setspace} \usepackage{listings} \usepackage{tocloft} \usepackage{epigraph} \usepackage{threeparttable} \usepackage{hyperref} % must be before cleveref \usepackage[capitalize]{cleveref} \hypersetup{ colorlinks=true, linkcolor=black, filecolor=black, citecolor=black, urlcolor=black, } \newcommand{\dmspaper}{Dwarshuis et al. Functionalized microcarriers improve T cell manufacturing by facilitating migratory memory T cell production and increasing CD4/CD8 ratio.~2019.~biorxiv.~https://doi.org/10.1101/646760} \newcommand{\modelpaper}{Odeh-Couvertier et al. Predicting T Cell Quality During Manufacturing Through an Artificial Intelligence-based Integrative Multi-Omics Analytical Platform.~2019.~biorxiv.~https://doi.org/10.1101/2021.05.05.442854} %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % my attempt to make MATLAB code look pretty \definecolor{dkgreen}{rgb}{0,0.6,0} \definecolor{gray}{rgb}{0.5,0.5,0.5} \definecolor{mauve}{rgb}{0.58,0,0.82} \lstset{frame=tb, language=Matlab, aboveskip=3mm, belowskip=3mm, showstringspaces=false, columns=flexible, basicstyle={\small\ttfamily}, numbers=none, numberstyle=\tiny\color{gray}, keywordstyle=\color{blue}, commentstyle=\color{dkgreen}, stringstyle=\color{mauve}, breaklines=true, breakatwhitespace=true, tabsize=3 } %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % benevolently force figures stay in their own subsection % % NOTE the placeins package only has a 'section' option which puts % floatbarriers after every \section call; this does the same for \subsection \makeatletter \AtBeginDocument{% \expandafter\renewcommand\expandafter\subsection\expandafter {\expandafter\@fb@secFB\subsection}% \newcommand\@fb@subsecFB{\FloatBarrier \gdef\@fb@afterHHook{\@fb@topbarrier \gdef\@fb@afterHHook{}}}% \g@addto@macro\@afterheading{\@fb@afterHHook}% \gdef\@fb@afterHHook{}% } \makeatother % ...also center them \makeatletter \g@addto@macro\@floatboxreset\centering \makeatother %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % header configuration % % NOTE glossary can't apparently be used in section header (even thought it % would be nice) \doublespacing{} \titleformat{\chapter}[block]{\filcenter\bfseries\Large} {\MakeUppercase{\chaptertitlename} \thechapter: }{0pt}{\uppercase} \titleformat{\section}[block]{\bfseries\large}{}{0pt}{\titlecap} \titleformat{\subsection}[block]{\itshape\large}{}{0pt}{\titlecap} \titleformat{\subsubsection}[runin]{\bfseries}{}{0pt}{\titlecap} \setlist[description]{font=$\bullet$~\textbf\normalfont} \renewcommand*{\contentsname}{TABLE OF CONTENTS} \renewcommand{\listfigurename}{LIST OF FIGURES} \renewcommand{\listtablename}{LIST OF TABLES} \renewcommand{\cfttoctitlefont}{\hspace*{\fill}\Large\bfseries} \renewcommand{\cftaftertoctitle}{\hspace*{\fill}} \renewcommand{\cftlottitlefont}{\hspace*{\fill}\Large\bfseries} \renewcommand{\cftafterlottitle}{\hspace*{\fill}} \renewcommand{\cftloftitlefont}{\hspace*{\fill}\Large\bfseries} \renewcommand{\cftafterloftitle}{\hspace*{\fill}} \setlength{\cftsubsecnumwidth}{0.55in} \setlength{\cftfignumwidth}{0.5in} \setlength{\cfttabnumwidth}{0.5in} %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % acronyms for the lazy % % adding as many as possible has the added benefit of making the thesis longer % and making me sound more sophisticated % the many flavors of T cells \newcommand{\tcellacronym}[4]{ \newacronym[shortplural={T\textsubscript{#2}#4 cells}]{#1}{T\textsubscript{#2}#4}{#3 T cell} } \newglossary[slg]{symbolslist}{syi}{syg}{Symbolslist} \makeglossaries \tcellacronym{tn}{n}{naive}{} \tcellacronym{tcm}{cm}{central memory}{} \tcellacronym{tscm}{scm}{stem-memory}{} \tcellacronym{tem}{em}{effector-memory}{} \tcellacronym{teff}{eff}{effector}{} \tcellacronym{treg}{reg}{regulatory}{} \tcellacronym{th}{h}{helper}{} \tcellacronym{tc}{c}{cytotoxic}{} \tcellacronym{th1}{h}{type 1 helper}{1} \tcellacronym{th2}{h}{type 2 helper}{2} \tcellacronym{th17}{h}{IL-17 helper}{17} \newacronym{bcaa}{BCAA}{branched-chain amino acid} \newacronym{til}{TIL}{tumor infiltrating lymphocyte} \newacronym{tcr}{TCR}{T cell receptor} \newacronym{act}{ACT}{adoptive cell therapies} \newacronym{qc}{QC}{quality control} \newacronym{car}{CAR}{chimeric antigen receptor} \newacronym[longplural={monoclonal antibodies}]{mab}{mAb}{monoclonal antibody} \newacronym{ecm}{ECM}{extracellular matrix} \newacronym{cqa}{CQA}{critical quality attribute} \newacronym{cpp}{CPP}{critical process parameter} \newacronym{dms}{DMS}{degradable microscaffold} \newacronym{doe}{DOE}{design of experiments} \newacronym{adoe}{ADOE}{adaptive design of experiments} \newacronym{gmp}{GMP}{Good Manufacturing Practices} \newacronym{cho}{CHO}{Chinese hamster ovary} \newacronym{all}{ALL}{acute lymphoblastic leukemia} \newacronym{cll}{CLL}{chronic lymphoblastic leukemia} \newacronym{pdms}{PDMS}{polydimethylsiloxane} \newacronym{dc}{DC}{dendritic cell} \newacronym{il}{IL}{interleukin} \newacronym{apc}{APC}{antigen presenting cell} \newacronym{mhc}{MHC}{major histocompatibility complex} \newacronym{elisa}{ELISA}{enzyme-linked immunosorbent assay} \newacronym{nmr}{NMR}{nuclear magnetic resonance} \newacronym{haba}{HABA}{4-hydroxyazobenene-2-carboxylic-acid} \newacronym{pbs}{PBS}{phosphate buffered saline} \newacronym{bca}{BCA}{bicinchoninic acid assay} \newacronym{bsa}{BSA}{bovine serum albumin} \newacronym{hsa}{HSA}{human serum albumin} \newacronym{stp}{STP}{streptavidin} \newacronym{stppe}{STP-PE}{streptavidin-phycoerythrin} \newacronym{snb}{SNB}{sulfo-nhs-biotin} \newacronym{cug}{CuG}{Cultispher G} \newacronym{cus}{CuS}{Cultispher S} \newacronym{pbmc}{PBMC}{peripheral blood mononuclear cells} \newacronym{macs}{MACS}{magnetic activated cell sorting} \newacronym{aopi}{AO/PI}{acridine orange/propidium iodide} \newacronym{igg}{IgG}{immunoglobulin G} \newacronym{pe}{PE}{phycoerythrin} \newacronym{fitc}{FITC}{Fluorescein} \newacronym{fitcbt}{FITC-BT}{Fluorescein-biotin} \newacronym{ptnl}{PTN-L}{Protein L} \newacronym{af647}{AF647}{Alexa Fluor 647} \newacronym{anova}{ANOVA}{analysis of variance} \newacronym{crispr}{CRISPR}{clustered regularly interspaced short palindromic repeats} \newacronym{mtt}{MTT}{3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} \newacronym{bmi}{BMI}{body mass index} \newacronym{a2b1}{A2B1}{integrin $\upalpha$1$\upbeta$1} \newacronym{a2b2}{A2B2}{integrin $\upalpha$1$\upbeta$2} \newacronym{nsg}{NSG}{NOD scid gamma} \newacronym{colb}{COL-B}{collagenase B} \newacronym{cold}{COL-D}{collagenase D} \newacronym{tsne}{tSNE}{t-stochastic neighbor embedding} \newacronym{umap}{UMAP}{uniform manifold approximation and projection} \newacronym{anv}{AXV}{Annexin-V} \newacronym{pi}{PI}{propidium iodide} \newacronym{rt}{RT}{room temperature} \newacronym{cas37}{Cas3/7}{Caspase-3/7} \newacronym{bcl2}{BCL-2}{B cell lymphoma 2} \newacronym{tmb}{TMB}{3,3',5,5'-Tetramethylbenzidine} \newacronym{gvhd}{GVHD}{graft-vs-host disease} \newacronym{bcma}{BCMA}{B-cell maturation antigen} \newacronym{di}{DI}{deionized} \newacronym{moi}{MOI}{multiplicity of infection} \newacronym{ifng}{IFN$\upgamma$}{interferon-$\upgamma$} \newacronym{tnfa}{TNF$\upalpha$}{tumor necrosis factor-$\upalpha$} \newacronym{gmcsf}{GM-CSF}{granulocyte-macrophage colony stimulating factor} \newacronym{sql}{SQL}{structured query language} \newacronym{fcs}{FCS}{flow cytometry standard} \newacronym{ivis}{IVIS}{in vivo imaging system} \newacronym{iacuc}{IACUC}{institutional animal care and use committee} \newacronym{hbss}{HBSS}{Hank's buffered saline solution} \newacronym{leaf}{LEAF}{low endotoxin, azide-free} \newacronym{cytof}{CyTOF}{cytometry time-of-flight} \newacronym{spade}{SPADE}{spanning-tree progression analysis of density-normalized events} \newacronym{ml}{ML}{machine learning} \newacronym{rf}{RF}{random forest} \newacronym{sr}{SR}{symbolic regression} \newacronym{gbm}{GBM}{gradient boosted trees} \newacronym{cif}{CIF}{conditional inference forests} \newacronym{lasso}{LASSO}{least absolute shrinkage and selection operator} \newacronym{svm}{SVM}{support vector machines} \newacronym{plsr}{PLSR}{partial least squares regression} \newacronym{mse}{MSE}{mean squared error} \newacronym{loocv}{LOO-CV}{leave-one-out cross validation} \newacronym{hsqc}{HSQC}{heteronuclear single quantum coherence} \newacronym{hla}{HLA}{human leukocyte antigen} \newacronym{zfn}{ZFN}{zinc-finger nuclease} \newacronym{talen}{TALEN}{transcription activator-like effector nuclease} \newacronym{qbd}{QbD}{quality-by-design} \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} \newacronym{scfv}{scFv}{single-chain fragment variable} \newacronym{hepes}{HEPES}{4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid} \newacronym{nhs}{NHS}{N-hydroxysulfosuccinimide} \newacronym{tocsy}{TOCSY}{total correlation spectroscopy} \newacronym{hplc}{HPLC}{high-performance liquid chromatography} \newacronym{grex}{G-Rex}{Gas Permeable Rapid Expansion} % symbols to make me sound mathier than I really am \newcommand{\evalat}[2]{#1\rvert_{#2}} \newcommand{\newsymbol}[3]{ \newglossaryentry{sym:#1}{name=\ensuremath{#2}, description={#3}, type=symbolslist} } \newsymbol{diff}{D}{diffusion coefficient of ligand} \newsymbol{appdiff}{D_{app}}{apparent diffusion coefficient of ligand} \newsymbol{geodiff}{\beta}{geometric diffusivity, which is a fractional parameter representing the tortuousity and void fraction of the microcarrier} \newsymbol{mcligconc}{C_{L,m}}{concentration of ligand in microcarrier} \newsymbol{bulkligconc}{C_{L,b}}{concentration of ligand outside microcarriers} \newsymbol{mcrecconc}{C_{R,m}}{concentration of receptor inside microcarriers} \newsymbol{flowrate}{Q}{molar flow rate of ligand} \newsymbol{mcflux}{N_{m}}{flux of ligand in microcarrier} \newsymbol{rad}{r}{radial position in the microcarrier} \newsymbol{interrad}{r_i}{radius of unbound:bound receptor interface in microcarriers} \newsymbol{intervol}{V_i}{volume of unbound:bound receptor interface in microcarriers} \newsymbol{mcrad}{R}{average radius of microcarriers} \newsymbol{mcnum}{n}{number of microcarriers in bulk} \newsymbol{vol}{V}{volume of bulk liquid in which microcarriers are suspended} \newsymbol{time}{t}{time} %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % SI units for uber nerds % NOTE the \SI macro is depreciated but the arch repo (!!!) hasn't been updated % with the latest package yet (texlive-science) \sisetup{per-mode=symbol,list-units=single} \DeclareSIUnit\IU{IU} \DeclareSIUnit\rpm{RPM} \DeclareSIUnit\carrier{carrier} \DeclareSIUnit\gauge{gauge} \DeclareSIUnit\dms{DMS} \DeclareSIUnit\stp{STP} \DeclareSIUnit\snb{SNB} \DeclareSIUnit\cell{cells} \DeclareSIUnit\ab{mAb} \DeclareSIUnit\normal{N} \DeclareSIUnit\molar{M} \DeclareSIUnit\mM{\milli\molar} \DeclareSIUnit\uM{\micro\molar} \DeclareSIUnit\gforce{\times{} g} %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % commands for lazy farts like me % gatech format conformity \newcommand{\mytitle}{ \Large{ \textbf{ Optimizing T Cell Manufacturing and Quality Using Functionalized Degradable Microscaffolds } } } \newcommand{\mycommitteemember}[3]{ \begin{flushleft} \noindent #1 \\ #2 \\ \textit{#3} \end{flushleft} } % a BME's best friend \newcommand{\invivo}{\textit{in vivo}} \newcommand{\invitro}{\textit{in vitro}} \newcommand{\exvivo}{\textit{ex vivo}} \newcommand{\Invivo}{\textit{In vivo}} \newcommand{\Invitro}{\textit{In vitro}} \newcommand{\Exvivo}{\textit{Ex vivo}} % various CD-whatever crap \newcommand{\cd}[1]{CD{#1}} \newcommand{\anti}[1]{anti-{#1}} \newcommand{\antih}[1]{anti-human {#1}} \newcommand{\antim}[1]{anti-mouse {#1}} \newcommand{\acd}[1]{\anti{\cd{#1}}} \newcommand{\ahcd}[1]{\antih{\cd{#1}}} \newcommand{\amcd}[1]{\antim{\cd{#1}}} \newcommand{\pos}[1]{#1+} \newcommand{\cdp}[1]{\pos{\cd{#1}}} \newcommand{\pcd}[1]{\cdp{#1}~\si{\percent}} \newcommand{\cdn}[1]{\cd{#1}-} \newcommand{\ptmem}{\cdp{62L}\pos{CCR7}} \newcommand{\ptmemp}{\ptmem{}~\si{\percent}} \newcommand{\pth}{\cdp{4}} \newcommand{\pthp}{\pth{}~\si{\percent}} \newcommand{\ptk}{\cdp{8}} \newcommand{\ptmemh}{\pth\ptmem} \newcommand{\ptmemk}{\ptk\ptmem} \newcommand{\dpthp}{$\Updelta$\pthp{}} \newcommand{\ptcar}{\gls{car}+} \newcommand{\ptcarp}{\ptcar~\si{\percent}} % so I don't need to worry about abbreviating all the different interleukins \newcommand{\il}[1]{\gls{il}-#1} \newcommand{\ilr}[1]{\gls{il}-#1R} % ...and this one is just plain annoying \newcommand{\ilXVra}[1]{\ilr{15}$\upalpha$} % DOE stuff I don't feel like typing ad-nauseam \newcommand{\pilII}{\il{2} concentration} \newcommand{\pdms}{\gls{dms} concentration} \newcommand{\pmab}{functional \gls{mab} surface density} \newcommand{\rmemh}{total \ptmemh{} cells} \newcommand{\rmemk}{total \ptmemk{} cells} \newcommand{\rratio}{CD4/CD8 ratio} % vendor and product stuff I don't feel like typing \newcommand{\catnum}[2]{(#1, #2)} \newcommand{\product}[3]{#1 \catnum{#2}{#3}} \newcommand{\thermo}{Thermo Fisher} \newcommand{\gehc}{GE Healthcare} \newcommand{\sigald}{Sigma Aldrich} \newcommand{\miltenyi}{Miltenyi Biotech} \newcommand{\bl}{Biolegend} \newcommand{\bd}{Becton Dickenson} \newcommand{\pltread}{BioTek plate reader} % the obligatory misc category \newcommand{\inlinecode}{\texttt} \newcommand{\subcap}[2]{\subref{#1}) #2} \newcommand{\sigkey}{Significance test key: *p<0.1; **p < 0.05; ***p<0.01} \newcommand{\nVI}{NALM-6} %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % ditto for environments \newenvironment{mytitlepage}{ \begin{singlespace} \begin{center} } { \end{center} \end{singlespace} } %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % begin document (proceed with caution) \begin{document} \pagenumbering{gobble} \begin{titlepage} \begin{mytitlepage} \mytitle{} \vfill \Large{ A Dissertation \\ Presented to \\ The Academic Faculty \\ \vspace{1.5em} by \vspace{1.5em} Nathan John Dwarshuis \\ \vfill In Partial Fulfillment \\ of the Requirements for the Degree \\ Doctor of Philosophy in Biomedical Engineering in the \\ Wallace H. Coulter Department of Biomedical Engineering \vfill Georgia Institute of Technology and Emory University \\ December 2021 \vfill COPYRIGHT \copyright{} BY NATHAN J. DWARSHUIS } \end{mytitlepage} \end{titlepage} \onecolumn \clearpage \begin{mytitlepage} \mytitle{} \end{mytitlepage} \vfill \large{ \noindent Committee Members \begin{multicols}{2} \begin{singlespace} \mycommitteemember{Dr.\ Krishnendu\ Roy\ (Advisor)} {Department of Biomedical Engineering} {Georgia Institute of Technology and Emory University} \vspace{1.5em} \mycommitteemember{Dr.\ Madhav\ Dhodapkar} {Department of Hematology and Medical Oncology} {Emory University} \vspace{1.5em} \mycommitteemember{Dr.\ Melissa\ Kemp} {Department of Biomedical Engineering} {Georgia Institute of Technology and Emory University} \columnbreak{} \null{} \vfill \mycommitteemember{Dr.\ Wilbur\ Lam} {Department of Biomedical Engineering} {Georgia Institute of Technology and Emory University} \vspace{1.5em} \mycommitteemember{Dr.\ Sakis\ Mantalaris} {Department of Biomedical Engineering} {Georgia Institute of Technology and Emory University} \end{singlespace} \end{multicols} \vspace{1.5em} \hfill Date Approved: } \clearpage \vspace*{\fill} \begin{center} You cannot answer a question that you cannot ask, and you cannot ask a question for which you have no words. \medskip \textit{Judea Pearl -- The Book of Why} \nocite{Pearl2018} \end{center} \vspace{1in} \vspace*{\fill} \clearpage \pagenumbering{roman} \chapter*{ACKNOWLEDGEMENTS} \addcontentsline{toc}{chapter}{ACKNOWLEDGMENTS} There are many people without which this work would not have been possible. Firstly, I would like to thank my advisor, Krish Roy, for his mentoring and guidance as well as his support in my exploring my own ideas throughout this project. In addition to the Roy lab as a whole, I should specifically recognize Dr's Ranjna Madan-Lala and Kyung-Ho Roh for writing the NSF EAGER grant that initially funded this work and providing the foundation of ideas, Dr Hannah Wilson Song for assisting me with much of the cell-based work, Dr Pallab Pradhan for assisting with the animal studies, and Miguel Armenta Ochoa and Ritika Jain for their assistance as well as their ideas for new directions of this work as they continue it beyond my tenure. I would also like to thank the undergraduates and high school students I had the pleasure of mentoring, Anokhi Patel, Kate Richardson, Zahra Mousavi Karimi, Sambhav Jain, and Lauren Bailey. Beyond the Roy lab, I should thank the Cell Manufacturing Technologies (CMaT) family at large, especially our collaborators Art Edison, Max Colonna, Wandaliz Torres-Garcia, Valerie Odeh-Couvertier, Theresa Kotanchek, and Bruce Levine. Additionally, I would like to thank the staff and faculty who fundamentally supported this work, including Andrea Soyland, Punya Mardhanan, Carol Mills, Carla Zachery, Adrienne Williams, Sommer Durham, Laxmi Krishnan, Andrew Shaw, Aaron Lifland, and Paramita Chatterjee. Finally, I would like to thank my friends and family for their unconditional support throughout this process, as well as Arch Enemy and Megadeth because reasons. \clearpage \tableofcontents \clearpage \listoftables \addcontentsline{toc}{chapter}{LIST OF TABLES} \clearpage \listoffigures \addcontentsline{toc}{chapter}{LIST OF FIGURES} \clearpage \printglossary[type=\acronymtype,title=LIST OF ABBREVIATIONS,toctitle=LIST OF ABBREVIATIONS,style=index] \clearpage \printglossary[type=symbolslist,title=LIST OF SYMBOLS,toctitle=LIST OF SYMBOLS, style=index] \clearpage \chapter*{summary} \addcontentsline{toc}{chapter}{SUMMARY} \Gls{act} using \gls{car} T cells have shown promise in treating cancer, but manufacturing large numbers of high quality cells remains challenging. Currently approved T cell expansion technologies involve \acd{3} and \acd{28} \glspl{mab}, usually mounted on magnetic beads. This method fails to recapitulate many key signals found \invivo{} and is also heavily licensed by a few companies, limiting its long-term usefulness to manufactures and clinicians. Furthermore, highly potent, anti-tumor T cells are generally less-differentiated subtypes such as \acrlongpl{tcm} and \acrlongpl{tscm}. Despite this understanding, little has been done to optimize T cell expansion for generating these subtypes, including measurement and feedback control strategies that are necessary for any modern manufacturing process. The goal of this dissertation was to develop a microcarrier-based \gls{dms} T cell expansion system and determine biologically-meaningful \glspl{cqa} and \glspl{cpp} that could be used to optimize for highly-potent T cells. In \cref{aim1}, we developed and characterized the \gls{dms} system, including \gls{qc} steps. We also demonstrated the feasibility of expanding high-quality T cells. In \cref{aim2a,aim2b}, we used \gls{doe} methodology 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 final product. In \cref{aim3}, we demonstrated the effectiveness of the \gls{dms} platform \invivo{}. This thesis lays the groundwork for a novel T cell expansion method which can be utilized at scale for clinical trials and beyond. \clearpage \pagenumbering{arabic} \chapter{INTRODUCTION} \section*{overview} 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 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, difficult-to-scale manufacturing process with little control on 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 \il{2}. 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. These include 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 activate 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 are 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. This dissertation describes a novel \acrlong{dms}-based method for expanding T cells using porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab}. Microcarriers have historically been used in 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 increase quantity and quality of T cells as compared to state-of-the-art bead-based expansion. We also hypothesized that such 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} The specific aims of this dissertation are outlined in \cref{fig:graphical_overview}. \begin{figure*}[ht!] \begingroup \includegraphics[width=\textwidth]{../figures/overview.png} \endgroup \caption[Project Overview]{High-level overview.} \label{fig:graphical_overview} \end{figure*} \subsection*{aim 1: develop and optimize a novel T cell expansion process that mimics key components of the lymph nodes} In this first aim, we demonstrated the process for manufacturing \glspl{dms}, including \gls{qc} steps that are necessary for translation of this platform into a scalable manufacturing setting. We also demonstrated that the \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 therapies. Finally, we showed \invitro{} that the \gls{dms} platform can be 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 accomplished through two sub-aims: \begin{itemize} \item[A --] Develop computational methods to control and predict T cell expansion and quality \item[B --] Perturb \gls{dms} expansion to identify additional mechanistic controls for expansion and quality \end{itemize} \subsection*{aim 3: confirm potency of T cells from novel T cell expansion process using \invivo{} xenograft mouse model} 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 \gls{all}. \section*{outline} 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 INNOVATION}\label{background} \section{Background} \subsection{Quality by Design in Cell Manufacturing} The challenges for the cell manufacturing field are significant. Unlike other industries which manufacture inanimate products such as automobiles and semiconductors, the cell manufacturing industry needs to contend with the fact that cells are living entities which can change with every process manipulation\cite{Kirouac2008, Little2006, Pirnay2012, Rousseau2013}. This is further compounded by the lack of standardization and limited regulation. In order to overcome these barriers, adopting a systemic approach to cell manufacturing using \acrfull{qbd} principles will be extremely important\cite{Kirouac2008}. In \gls{qbd}, the objective is to reproducibly manufacturing products which minimizes risk for downstream stakeholders (in this case, the patient). This entails determining \acrlongpl{cqa} and \acrlongpl{cpp} and incorporating them into models which can explain and predict the cell manufacturing process. \Glspl{cqa} are measurable properties of the product that are used to define its functionality and hence quality. \glspl{cqa} are important for defining the characteristics of a ``good'' product (release criteria) but also for ensuring that a process is on track to making such a product (process control). In the space of cell manufacturing, examples of \glspl{cqa} include markers on the surface of cells and readouts from functional assays such as killing assays. In general, these are poorly understood if they exist at all. \glspl{cpp} are parameters which may be tuned to control the outcome of process and the quality of the final product. Examples include the type of media used and the amount of \il{2} added. While these can be easy to control, the effect they have on the final outcome is generally unknown. Once \glspl{cpp} are known, they can be optimized to ensure that costs are minimized and potency of the cellular product is maximized. The topic of discovering novel \glspl{cpp} and \glspl{cqa} in the context of this work are discussed further in \cref{sec:background_doe} and \cref{sec:background_quality}/\cref{sec:background_cqa} respectively. \subsection{T Cells for Immunotherapies} A variety of T cell therapies have been utilized with varying degrees of success, and we describe a few of the most prominent below. We should note that while this work focuses on the application of \gls{car} T cell therapies, in theory the technology developed in this dissertation could theoretically apply to any T cell-based therapy with little to no modification. One of the first successful T cell-based immunotherapies against cancer is \glspl{til}\cite{Rosenberg2015}. This method works by excising tumor fragments from a patient, allowing the tumor-reactive lymphocytes to expand \exvivo{} from within these fragments, and then administered these lymphocytes back to the patient along with a high dose of \\il{2}\cite{Rosenberg1988}. In particular, \gls{til} therapy has shown robust results in treating 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, Santoiemma2015}, and their presence is generally associate with favorable outcomes\cite{Clark1989}. \glspl{til} are heterogeneous cell mixtures and generally are comprised of CD3 T cells and $\upgamma\updelta$ T cells\cite{Nishimura1999, Cordova2012}. To date, there are over 250 open clinical trials using \glspl{til}. Besides \glspl{til}, the other broad class of T cell immunotherapies that has achieved great success in treating cancer in recent decades are gene-modified T cells. Rather than expand T cells that are present natively (as is the case with \gls{til} therapy), gene-modified T cell therapies entail extracting T cells from either the cancer patient (autologous) or a healthy donor (allogeneic) and reprogramming them genetically to target a tumor antigen (see \cref{sec:background_source} for an overview of how T cells can be sourced). This approach offers much more flexibility, as the degree of reprogramming is only limited by the scale and possibilities of gene-editing technology, which has rapidly accelerated in recent decades\cite{Rosenberg2015}. T cells with transduced \glspl{tcr} were first designed to overcome the limitations of \glspl{til}\cite{Rosenberg2015, Wang2014}. In this case, T cells are transduced \exvivo{} with a lentiviral vector to express a \gls{tcr} targeting a tumor antigen. T cells transduced with \glspl{tcr} have shown robust results against melanoma \cite{Robbins2011}, synovial sarcoma\cite{Morgan2006}, and others\cite{Ikeda2016}. To date, there are over 200 clinical trials using T cells with transduced \glspl{tcr}. While transduced \glspl{tcr} offer some flexibility in retargeting T cells toward relevant tumor antigens, they are still limited in that they can only target antigens that are presented via \gls{mhc}. \Acrlong{car} T cells overcome this limitation by linking a \gls{tcr}-independent antigen recognition domain with the stimulatory and costimulatory machinery of a T cell. \gls{car} T cells were first demonstrated in 1989, where the authors swapped the antigen-recognition domains of a native \gls{tcr} with a that of a foreign \gls{tcr}\cite{Gross1989}. Since then, this method has progressed to using an \gls{scfv} for antigen recognition, a CD3$\upzeta$ domain for the stimulatory signal, and a CD28, OX-40, or 4-1BB domains for the costimulatory signal. Since these can all be expressed with one protein sequence, \gls{car} T cells are relatively simple to produce and require only a single genetic transduction step (usually a lentiviral vector) to reprogram a batch T cells \exvivo{} toward the desired antigen. \gls{car} T cells have primarily found success in against CD19- and CD20-expressing tumors such as \gls{all} and \gls{cll} (eg B-cell malignancies)\cite{Kalos2011, Brentjens2011, Kochenderfer2010, Maude2014, Till2012, Till2008}. Out of all the T cell therapies discussed thus far, \gls{car} T cells have experienced the most commercial success and excitement. In 2017, Novartis and Kite Pharma acquired FDA approval for \textit{Kymriah} and \textit{Yescarta} respectively, both of which are \gls{car} T cell therapies against B-cell malignancies. \gls{car} T cells are under further exploration for use in many other tumors, including multiple myeloma, mesothelioma, pancreatic cancer, glioblastoma, neuroblastoma, and prostate cancer, breast cancer, non-small-cell lung cancer, and others\cite{Rosenberg2015, Wang2014, Fesnak2016, Guo2016}. To date, there are almost 1000 clinical trials using \gls{car} T cells. \subsection{Scaling T Cell Expansion} In order to scale T cell therapies, automation and bioreactors will be necessary. To this end, several choices have found success in the clinic. The WAVE bioreactor (GE Healthcare) is the choice of expansion for many clinical applications\cite{Brentjens2011, Hollyman2009, Brentjens2013}. It is part of a broader class of bioreactors that consist of rocking platforms that agitate a bag filled with media and cells. Importantly, it has built-in sensors for measuring media flow rate, \ce{CO2}, \ce{O2}, pH, and nutrient consumption which enables automation. Unfortunately, in some settings this is not considered scalable as only one bag per bioreactor is allowed at once\cite{Roddie2019}. The other disadvantage with the WAVE system is that it keeps cells far apart by design, which could have negative impact on cross-talk, activation, and growth\cite{Somerville2012}. % BACKGROUND find clinical trials (if any) that use this Alternatively, the CliniMACS Prodigy (Miltenyi) is an all-in-one, fully-closed system that removes the need for expensive cleanrooms and associated personnel\cite{Kaiser2015, Bunos2015}. It contains modules to perform transduction, expansion, and washing. This setup is less prone to mistakes, since most steps are internal to the machine. Initial investigations have shown that it can yield T cells doses required for clinical use\cite{Zhu2018}. At the time of writing, several clinical trial are underway which use the CliniMACS, although mostly for stem-cell based cell treatments. Finally, another option that has been investigated for T cell expansion is the \gls{grex} bioreactor (Wilson Wolf). This is effectively a tall tissue-culture plate with a porous membrane at the bottom. This allows large volumes of media to be loaded without suffocating the cells, which can exchange gas through the membrane. While this is quite similar to plates and flasks normally used for small-scale research, the important difference is that its larger size requires fewer interactions and keeps the cells at a higher nutrient concentration for longer periods of time. However, it is still a an open system and requires manual (by default) interaction from an operator to load, feed, and harvest the cell product. \gls{grex} bioreactors have been using to grow \glspl{til}\cite{Jin2012} and virus-specific T cells\cite{Gerdemann2011}. 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 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} T cells for cell manufacturing can be obtained broadly via two paradigms: autologous and allogeneic. The former involves obtaining T cells from a patient and giving them back to the same patient after \exvivo{} expansion and genetic modification. The latter involves taking T cells from a healthy donor, expanding them and manipulating them as desired, storing them long term, and then giving them to multiple patients. There are advantages and disadvantages to both, and in some cases such as \gls{til} therapy, the only option is to use autologous therapy. Autologous T cells by default are much safer. By definition, they will have no cross-reactivity with the patient and thus \gls{gvhd} is not a concern\cite{Decker2012}. However, there are numerous disadvantages. Autologous therapies are over 20 times more costly as the process needs to be repeated for every patient\cite{Harrison2019}. Compounding this, many cell products are manufactured at a centralized location, so cells need to be shipped on dry ice from the hospital and back. This adds days to the process, which is critical for patients with fast moving diseases. Manufacturing could be done on-site in a decentralized manner, but this requires more equipment and personnel overall. Sourcing cells from a diseased patient has many drawbacks in itself. Cancer patients (especially those with chronic illnesses) often have exhausted T cells which expand far less readily and are consequently less potent\cite{Wherry2015, Ando2019, Zheng2017}. Additionally, they may have high frequencies of \glspl{treg} which have an inhibitory effect on immunotherapies\cite{Decker2012}. Removing these cells as well as purifying \glspl{th1} may enhance the potency of the final product\cite{Goldstein2012, Drela2004, Rankin2003, Luheshi2013, Grotz2015}; however, this makes the overall process more expensive as an additional separation step is required. Allogeneic T cell therapies overcome nearly all of these disadvantages. Donor \glspl{pbmc} are easy to obtain, they can be processed in centralized locations, and they can be stored easily under liquid nitrogen. Donors can also be screened to find those with optimal anti-tumor cells. The key is overcoming \gls{gvhd}. Obviously this could be done analogously to transplants where patients find a ``match'' for their \gls{hla} type, but this severally limits options. This can be overcome by using advanced gene-editing tools (eg \glspl{zfn}, \glspl{talen}, or \gls{crispr}) to remove the native \gls{tcr} and thus prevent the donor T cells from attacking host tissue\cite{Liu2019, Wiebking2020, Provasi2012, Berdien2014, Themeli2015}. This obviously complicates the process, as additional edits besides the insertion of the \gls{car} would be required, and these technologies are not yet very efficient. To date there are about 10 open clinical trials utilizing allogeneic T cell therapies edited with \gls{crispr} to reduce the likelihood of \gls{gvhd}. \subsection{Overview of T Cell Quality}\label{sec:background_quality} T cells are highly heterogeneous and can exist in a variety of states and subtypes, many of which can be measured (at least indirectly) though biomarkers such as cell surface proteins. Identifying and understanding these biomarkers are the basis for \glspl{cqa} which can be used to for process control, release criteria, and initial cell source screening. 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 thymus) as \glspl{tn}. When they become activated in the secondary lymphoid organs, they differentiate from \gls{tn} to \glspl{tscm}, \glspl{tcm}, \glspl{tem}, and finally \glspl{teff}\cite{Gattinoni2012}. Subtypes earlier in this process are generally called ``memory'' or ``memory-like'' cells (eg \gls{tscm} and \gls{tcm}), and have been shown to have increased potency toward a variety of tumors, presumably due to their higher capacity for self-renewal and replication, enhanced migratory capacity, and/or increased engraftment potential\cite{Xu2014, Gattinoni2012, Fraietta2018, Gattinoni2011, Turtle2009}. 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 create more durable responses\cite{Donia2012}. Additionally, clonal diversity decreases following the infusion of \gls{car} T cell therapies, which demonstrates that only a few clones are self-renewing and therefore responsible for the overall response\cite{Sheih2020}. Memory T cells can be quantified easily using surface markers such as CD62L, CCR7, CD27, CD45RA, and CD45RO. Furthermore, memory markers are inversely related to exhaustion markers which are negatively associated with clinical outcomes\cite{Lee2013}. These cells in particular are seen in patients with chronic immune activation such as patients with chronic cancers. 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 cytokines which coordinate the immune response while CD8+ \glspl{tc} are responsible for killing tumors or infected cells using specialized lytic enzymes. Since \glspl{tc} actually possess the killing function, it seems intuitive that \glspl{tc} would be most important for anti-tumor immunotherapies. However, in mouse models with glioblastoma, survival was negatively impacted when \glspl{th} were removed\cite{Wang2018}. Furthermore, \glspl{th} have been shown to have cytotoxic properties on their own and also show resistance to exhaustion compared to \glspl{tc}\cite{Yang2017}. While T cell products with a defined ratio of CD4 and CD8 T cells have been utilized, they are more expensive than products with undefined ratios as the T cells need to be sorted and recombined, adding additional complexity\cite{Turtle2016}. While less of a focus in this dissertation, other quality markers exists to assess the overall killing potential and safety of the T cell product. Numerous methods exists to detect the killing capacity of \gls{car} T cells, many of which involve either measuring the lysis of a target cell using a dye or a 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 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 assay to detect metabolism. Finally, for safety, retro- or lentivirally transduced T cell products must be tested for replication competent vectors\cite{Wang2013}, and all cell products in general should be tested for bacterial or fungal contamination. \subsection{T Cell Activation Methods}\label{sec:background_activation} In order for T cells to be expanded \exvivo{} they must be activated with a stimulatory signal (Signal 1) and a costimulatory signal (Signal 2). \Invivo{}, Signal 1 is administered via the \gls{tcr} and the CD3 receptor when \glspl{apc} 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 \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 many other potential signals present. T cells have other receptors such as OX40, 4-1BB, and ICOS which are costimulatory along with CD28, and \glspl{apc} have corresponding ligands for these depending on the nature of the pathogen they have detected\cite{Azuma2019}. Furthermore, T cells exist in high cell density within the secondary lymphoid organs, which allows efficient cytokine cross-talk in an autocrine and paracrine manner. These cytokines are responsible for triggering proliferation (in the case of \il{2}) and subset differentiation (in the case of many others)\cite{Luckheeram2012}. By tuning the signal strength and duration of Signal 1, Signal 2, the various costimulatory signals, and the cytokine milieu, a variety of T cell phenotypes can be actualized. There are many ways to activate T cells \invitro{}, but the simplest and most common is to use \glspl{mab} that cross-link CD3 and CD28, which supply Signal 1 and Signal 2 without the need for antigen (which also means all T cells are activated and not just a few specific clones). Additional signals may be supplied in the form of cytokines (eg \il{2}, \il{7}, or \il{15}) or feeder cells\cite{Forget2014}. 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, Piscopo2017, Roddie2019, Bashour2015}. The simplest is to use \acd{3} and \acd{28} \glspl{mab} bound to a 2D surface such as a plate, and this can be 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 distinct method along these lines is to use multivalent activators such as ImmunoCult (StemCell Technologies) or Expamer (Juno Therapeutics) which have increased cross-linking capacity compared to traditional \glspl{mab}. Beyond 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 MACSbeads (\miltenyi{}), which are easy to separate using magnetic washing plates. Magnetic nanobeads such as TransAct (\miltenyi{}) work by a similar principle except they can be removed via centrifugation rather than a magnetic washing plate. Cloudz (RnD Systems) are another bead-based T cell expansion which mounts \acd{3} and \acd{28} \glspl{mab} on alginate microspheres, which are not only easily degradable but are also softer, which can have a positive impact on T cell activation and phenotype\cite{Lambert2017, OConnor2012}. A problem with all of these commercial solutions is that they only focus on 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 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 \glspl{dc}\cite{Forget2014}. While this can theoretically mimic many components of the lymph node, it is hard to scale due to the complexity and inherent variability of using cell lines in a \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{}. None of these have been shown to expand high quality T cells as outlined in \cref{sec:background_quality}. \subsection{Microcarriers in Bioprocessing} In this work, we explored microcarriers as the basis for an alternative to the methods described in \cref{sec:background_activation}. 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 grow adherent cells ``in suspension,'' effectively turning a 2D flask into a 3D culture system\cite{WEZEL1967}. Microcarriers are generally spherical and are several hundred \si{\um} in diameter, which means they collectively have a much higher surface area than a traditional flask when matched for volume. Consequently, this means that microcarrier-based cultures can operate with much lower footprints than flask-like systems. Microcarriers also allow cell cultures to operate more like traditional chemical engineering processes, wherein a \gls{cstr} may be employed to enhance oxygen transfer, maintain pH, and continuously supply nutrients\cite{Derakhti2019}. A variety of microcarriers have been designed, primarily differing in their choice of material and macroporous structure. Key concerns driving these choices have been cell attachment at the beginning of culture and cell detachment at the harvesting step\cite{Derakhti2019}. Many microcarriers simply use polystyrene (the material used for tissue culture flasks and dishes in general) with no modification (SoloHill Plastic, Nunc Biosilon), with a cationic surface charge (SoloHill Hillex) or coated with an \gls{ecm} protein such as collagen (SoloHill Fact III). Other base materials have been used such as dextran (GE Healthcare Cytodex), cellulose (GE Healthcare Cytopore), and glass (\sigald{} G2767), all with similar surface modifications (if any). Additionally, some microcarriers such as \gls{cus} and \gls{cug} are composed entirely out of protein (in these cases, porcine collagen) which also allows the microcarriers to be enzymatically degraded. In the case of non-protein materials, cells may still be detached using enzymes but these require similar methods to those currently used in flasks such as trypsin which target the cellular \gls{ecm} directly. Since trypsin and related enzymes tends to be harsh on cells, an advantage of using entirely protein-based microcarriers is that they can be degraded using a much safer enzyme such as collagenase, at the cost of being more expensive and also being harder to make \gls{gmp}-compliant\cite{Derakhti2019}. Going one step further, some microcarriers are composed of a naturally degrading scaffold such as alginate, which do not need an enzyme for degradation. Finally, microcarriers can differ in their overall structure. \gls{cug} and \gls{cus} microcarriers as well as the Cytopore microcarriers are macroporous, meaning they have a porous network in which cells can attach throughout their interior. This drastically increases the effective surface area and consequently the number of cells which may be grown per unit volume. Other microcarriers are microporous (eg only permeable to small molecules) or not porous at all; in either case, cells can only grow on the outer surface. Microcarriers have been mainly used for 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 therapies in the context of regenerative medicine\cite{Zhang2016, Saltz2016, Park2013, Malda2006}. However, all these cell types 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. Their protein-based composition makes functionalization easy; the surface is rich in lysine residues which can be easily bonded with a base-reactive linker such as \gls{snb}. 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{Integrins and T Cell Signaling} Because the microcarriers used in this work are derived from collagen, one key question is how these collagen domains may interact with the T cells during culture. This question is further explored in \cref{aim2b}. 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 interact with collagen fibers in the lymph node as the collagen fibers are sheathed with stromal fibroblasts\cite{Dustin2001, Ebnet1996}. However, the \gls{ecm} of peripheral tissues is dense where exposed collagen fibers are available to interact with T cells. Furthermore, T cells have been shown \invitro{} to crawl along collagen fibers in the presence of \glspl{apc}, facilitating short encounters with \glspl{apc}\cite{Gunzer2000}. While this may not be ideal \invivo{} due to the lack of cumulative signal received by \glspl{apc}\cite{Dustin2001}, it may be advantageous to include collagen domains \invitro{} as the mode of activation is not specific to any given clone. The major surface receptors for collagen are \gls{a2b1} and \gls{a2b2}\cite{Dustin2001, Hemler1990}. These receptors are not expressed on naive \gls{tn} cells and thus presence and stimulation of collagen alone is not sufficient for activation or expansion\cite{Hemler1990}; however, they have been shown to possess many functions that may be useful for T cell expansion. First, they can act in a costimulatory manner which leads to increased proliferation\cite{Rao2000}. Furthermore, \gls{a2b1} and \gls{a2b2} seem to protect Jurkat cells against Fas-mediated apoptosis in the presence of collagen I\cite{Aoudjit2000, Gendron2003}. Finally, these receptors can increase \gls{ifng} production \invitro{} when T cells derived from human \glspl{pbmc} are stimulated in the presence of collagen I\cite{Boisvert2007}. \subsection{The Role of IL15 in Memory T Cell Proliferation} \il{15} is a cytokine that is involved with the proliferation and homeostasis of memory T cells. Its role in the work of this dissertation is the subject of further exploration in \cref{aim2b}. Functionally, mice lacking the gene for either \il{15}\cite{Kennedy2000} or its high affinity receptor \ilXVra{}\cite{Lodolce1998} are generally healthy but show a deficit in memory CD8 T cells, thus underscoring this cytokine's importance in producing memory T cells for immunotherapies. T cells themselves express \il{15} and all of its receptor components\cite{MirandaCarus2005}. Additionally, blocking \il{15} itself or \ilXVra{} \invitro{} has been shown to inhibit homeostatic proliferation of resting human T cells\cite{MirandaCarus2005}. \il{15} has been puzzling historically because it shares almost the same pathway as \il{2} yet has different functions\cite{Stonier2010, Osinalde2014, Giri1994, Giri1995}. In particular, both cytokines bond with heterotrimeric receptors which share the common $\upgamma$ subchain (CD132) as well as the \il{2} $\upbeta$ receptor (CD122). The difference is the third subchain which is either the \il{2} $\upalpha$ receptor (CD25) or the \il{15} $\upalpha$ chain respectively, both of which have high affinity for their respective ligands. The \ilXVra{} chain itself does not have any signaling capacity, and therefore all the signaling resulting from \il{2} is mediated thought the $\upbeta$ and $\upgamma$ chains (namely via JAK1 and JAK3, which leads to STAT5 activation, which leads to T cell activation). \ilXVra{} itself has some innate signaling capacity, but this is poorly characterized in lymphocytes\cite{Stonier2010}. Thus there is a significant overlap between the functions of \il{2} and \il{15} due to their receptors sharing the $\upbeta$ and $\upgamma$ chains, and perhaps the main driver of their differential functions 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 from being membrane-bound to its receptor\cite{Stonier2010}. Particularly, \ilXVra{} binds to soluble \il{15} which produces a complex that can transmit signals to close neighboring cells (so called \textit{trans} presentation). This has been demonstrated in adoptive cell models, where T cells lacking \ilXVra{} were able to generate memory T cells and proliferate only when other cells were present which expressed \ilXVra{} \cite{Burkett2003, Schluns2004}. The implication of this mechanism is that cells expression \ilXVra{} either need to express \il{15} themselves or be near other cells expressing \il{15}, and other cells 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} manner, where \ilXVra{}/\il{15} complexes may bind to the $\upbeta$ and $\upgamma$ chains on the same cell, assuming each subchain is expressed and soluble \il{15} is available\cite{Olsen2007}. Finally, \ilXVra{} itself can exist in soluble form, which can bind to \il{15} and signal to cells which are not adjacent to the source independent of the \textit{cis/trans} mechanisms already described\cite{Budagian2004}. \subsection{Overview of Design of Experiments}\label{sec:background_doe} The \gls{dms} system described in this dissertation has many parameters that can be optimized and controlled (eg \glspl{cpp}). A \gls{doe} is an ideal framework to test multiple parameters simultaneously and determine which are relevant \glspl{cpp}. 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 throughout the \nth{20} century such as the automotive and semiconductor industries where engineers needed to minimize downtime and resource consumption on full-scale production lines. At its core, a \gls{doe} is simply a matrix of conditions to test where each row (usually called a ``run,'' which is the term used throughout this work) corresponds to one experimental unit for which the conditions are applied, and each column represents a parameter of concern to be tested. The values in each cell represent the level of each parameter. When the experiment is performed using this matrix of conditions, the results are be summarized into one or more ``responses'' that correspond to each run. These responses are then be modeled (usually using linear regression) to determine the statistical relationship (also called an ``effect'') between each parameter and the response(s). 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 \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 of the parameters and the goal of the experimenter, they all share common principles: \begin{description} \item [randomization --] The order in which the runs are performed should be randomized. This is to guarantee that the tested parameters are independent of any unobserved influences to the response, and thus allows the causal effect of each parameter to be isolated completely\footnote{this is why \glspl{doe} are sometimes called ``black box models;'' one can can safely say ``this parameter causes that'' without paying attention to the causal structure of 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 statistical, replicates should be used to ensure that the underlying distribution of errors can be estimated. While this is not strictly necessary to conclude results using a \gls{doe}, failure to use replications requires strong assumptions about the model structure (particularly in the case of 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 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 concerns the number of runs required to estimate a certain effect size. \item [orthogonality --] Orthogonality refers to the independence of each parameter in the design matrix. In other words, the levels tested in any given parameter add mutually-exclusive information about the response(s). Again, while not strictly necessary, orthogonality drastically simplifies the analysis of the experiment by allowing each parameter to be treated separately. In cases where orthogonality is impossible (which is often true in experiments with many categorical variables) strategies exist to maximize orthogonality. \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 relevant to the goals of the experiment but nonetheless could affect the response. A key assumption that is (usually) made in the case of blocking is that there is no interaction between the blocking variable and any of the experimental parameters. For example, in T cell expansion, if media lot were a blocking variable and expansion method were a parameter, we would by default assume that the effect of the expansion method does not depend on the media lot (even if the media lot itself might change the mean of the response). \end{description} \Glspl{doe} served three purposes in this dissertation. First, we used them as screening tools for potential \glspl{cpp}, which allowed us to test many input parameters and filter out the few that likely have the greatest effect on the response. Second, they were used to make a robust response surface model to predict optimums using relatively few resources, especially compared to full factorial or one-factor-at-a-time approaches. Third, we used \glspl{doe} to discover novel effects and interactions that generated hypotheses that could influence the directions for future work. To this end, the types of \glspl{doe} we generally used were fractional factorial designs with three levels, which enable the estimation of both main effects and second order quadratic effects. While there are advantages of using \glspl{doe}, it is important to recognize that they are not necessary or recommended for all experimental aims. In particular, \glspl{doe} excel when multiple factors (possible with multiple levels) need to be investigated at once and with a known degree of power. This is especially important when interaction is expected or needs to be 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 \gls{doe} (particularly a lower-powered screening experiment such as a resolution III design) with these factors and a few others may not be productive, and one is better off performing a few extra pilot experiments before doing a more complex design such as a central composite if desired. Furthermore, it should be noted that while the goal of a \gls{doe} is to minimize resources, the size necessary to justify a \gls{doe} may not be worth the experimental return. For biological work (or any domain with little automation), performing a randomized experiment with 20 to 30 runs is not trivial from a logistical perspective, especially when considering the number of manual manipulations and the chance of human error. 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 often not employed (and also not explicitly mentioned in papers). Assuming the experiment is manual, the largest reason to avoid randomization is that the experimentalist has no pattern to follow when administering treatment (such as ``add X to row 1 in well plate''), thus cognitive burden and the likelihood of mistakes increases. While \glspl{doe} are usually bigger with more parameters, the one-factor-at-a-time experiment usually performed in biological disciplines is much smaller and only has a few parameters, thus these concerns are minimal. There is no reason to 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 CQAs}\label{sec:background_cqa} % BACKGROUND at least attempt to show that there is alot of work in the space % identifying signaling networks In the context of T cell manufacturing, ideally we would have a set of non-destructive biomarkers that could both identify functional T cells and predict when a process is on track to deliver such functional T cells. T cells 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}. The complexity of these pathways dictates that we take a big-data approach to this problem. To this end, there are several multi-omic (or simply ``omic'') techniques that can be used to collect such datasets, which can then be fit to relevent responses (such as an endpoint quantification of memory T cells) to identify pertinent \glspl{cqa}. An overview of the techniques used in this work are: \begin{description} \item[luminex --] This is a multiplexed bead-based assay similar to \gls{elisa} that can measure many bulk (not single cell) cytokine concentrations simultaneously in a media sample. This is a destructive assay but does not require cells to obtain a measurement. \item[\gls{nmr} --] It is well known that T cells of different lineages have different metabolic profiles; for instance memory T cells have larger aerobic capacity and fatty acid 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 an attractive method that could be used for inline, real-time monitoring. \item[flow and mass cytometry --] Flow cytometry using fluorophores has been used extensively for immune cell analysis, but has a practical limit of approximately 18 colors\cite{Spitzer2016}. Mass cytometry is analogous to traditional flow cytometry except that it uses heavy-metal \gls{mab} conjugates, which has a practical limit of over 50 markers. While mass cytometry is less practical than simple flow cytometers such as the BD Accuri, we may find that only a few markers are required to accurately predict performance, and thus this could easily translate to industry using relatively cost-effective equipment. Both of these destructively analyze the cells themselves, but they have the advantage in that they are measuring a direct property of the cells and not a secreted product. \end{description} % BACKGROUND what about ssRNAseq? Upon collecting these omic datasets, determining the \glspl{cqa} becomes a computational problem. Predictions of the final product using data collected earlier in time can be made using any number of supervised learning techniques (linear and non-linear regression in all its forms) which in turn can be used to develop process control models. Unsupervised learning and dimensionality reduction techniques such as \gls{tsne}, \gls{umap}, and \gls{spade}\cite{Qiu2011, Qiu2017} can be performed to delineate clusters of interesting cell types and the markers that define them. Ultimately, identifying \glspl{cqa} will likely be an iterative process, wherein putative \glspl{cqa} will be identified, the corresponding \glspl{cpp} will be set to maximize high-quality products, and then additional data will be collected in the clinic as the product is tested on various patients with different indications. Additional \glspl{cqa} may be identified which better predict specific clinical outcomes, which can be fed back into the process model and optimized again. \section{Innovation} Several aspects of the \gls{dms} platform described in this dissertation are novel considering the state-of-the-art technology for T cell manufacturing: \begin{itemize} \item \Glspl{dms} offers a compelling alternative to state-of-the-art magnetic bead technologies (e.g. DynaBeads, MACS-Beads), which is noteworthy because the licenses for these techniques are controlled by only a few companies (Invitrogen and Miltenyi respectively). Because of this, bead-based expansion is more expensive to implement and therefore hinders companies from entering the rapidly growing T cell manufacturing arena. Providing an alternative will provide more options for manufacturers, leading to increased competition, lower costs, and higher innovation in the T cell manufacturing space. \item This is the first technology for T cell immunotherapies that selectively expands memory T cell populations with greater efficiency relative to bead-based expansion. Others have demonstrated methods that can achieve greater expansion of T cells, but not necessarily specific populations that are known to be potent. \item We used \glspl{doe} to discover and validate novel \glspl{cpp}, which is a strategy commonly used in non-biological industries but has yet to gain wide 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 the development of cell therapies, not only for process optimization but also hypotheses generation. Furthermore, it is a natural strategy to use even at small scale, where the cost of reagents, cells, and materials often precludes large sample sizes. \item The \gls{dms} system is be compatible with static bioreactors such as the \gls{grex} which has been adopted throughout the cell therapy industry. Thus this technology can be easily incorporated into existing cell therapy process that are performed at scale. \item We analyzed our system using a multiomics approach, which will enable the discovery of novel biomarkers to be used as \glspl{cqa}. While this approach has been applied to T cells previously, it has not been done in the context of a large \gls{doe}-based model. This approach is aware of the whole design space, and thus enables greater understanding of process parameters and their effect on cell phenotype. \end{itemize} \chapter{AIM 1}\label{aim1} \section{Introduction} This aim was to develop a functionalized microcarrier system that mimics several key aspects of the \invivo{} lymph node microenvironment. We compared compare this system to state-of-the-art T cell activation technologies for both expansion potential and memory cell formation. The governing hypothesis was that microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} will provide superior expansion and memory phenotype compared to state-of-the-art bead-based T cell expansion technology\footnote{adapted from \dmspaper{}}. \section{Methods} \subsection{DMS Functionalization}\label{sec:dms_fab} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/dms_flowchart.png} \endgroup \caption[\Acrshort{dms} Manufacturin Flowchart] {Overview of \gls{dms} manufacturing process.} \label{fig:dms_flowchart} \end{figure*} \product{\gls{cus}}{\gehc}{DG-2001-OO} or \product{\gls{cug}}{\gehc}{DG-0001-OO} were suspended at \SI{20}{\mg\per\ml} in 1X \gls{pbs} in a 15, 50, or 250 \si{\ml} conical tube. The mass of the tube with the \gls{pbs} and microcarriers were recorded to the nearest millimeter (subsequently referred to here as ``reaction mass''). The tube was centrifuged for \SI{30}{\second} at \SI{4500}{\gforce} to ensure all microcarriers were at the bottom of the tube. The tube was then autoclaved using a \SI{15}{\minute} cycle at \SI{121}{\degreeCelsius} and \SI{100}{\kPa\of{\gauge}}. All subsequent steps were done aseptically, and all reactions were carried out at \SI{20}{\mg\of{\carrier}\per\ml} at room temperature and agitated using an orbital shaker with a \SI{3}{\mm} orbit diameter. After autoclaving, the microcarriers were washed using sterile \gls{pbs} three times in a 10:1 volume ratio. The volume after these washes was corrected by massing the tube and its contents and adding or removing \gls{pbs} until the ``reaction mass'' was reached. \product{\Gls{snb}}{\thermo}{21217} was dissolved at approximately \SI{10}{\uM} in sterile ultrapure water, and the true concentration was then determined using the \gls{haba} assay (see below). \SI{2.5}{\nmol\of{\snb}\per\mg\of{\carrier}} (unless otherwise noted) was added to carrier suspension and allowed to react for \SI{60}{\minute} at \SI{700}{\rpm} of agitation. After the reaction, the amount of biotin attached to the microcarriers was determined indirectly by measuring the biotin in solution via the \gls{haba} assay (see below). The carriers were then washed three times, which entailed adding sterile \gls{pbs} in a 10:1 volumetric ratio, agitating at \SI{900}{\rpm} for \SI{10}{\minute}, adding up to a 15:1 volumetric ratio (relative to reaction volume) of sterile \gls{pbs}, centrifuging at \SI{1000}{\gforce} for \SI{1}{\minute}, and removing all liquid back down to the reaction volume. The volume of the \gls{pbs} was corrected by massing the tube and its contents and adding or removing \gls{pbs} until the tube mass matched the ``reaction mass.'' To coat the microcarriers with \product{\gls{stp}}{Jackson Immunoresearch}{016-000-114}, \SI{2}{\ug\of{\stp}\per\mg\of{\carrier}} was added and allowed to react for \SI{60}{\minute} at \SI{700}{RPM} of agitation. After the reaction, \SI{400}{\ul} supernatant (regardless of tube size) was taken for the \product{\gls{bca} assay}{\thermo}{23225} in order to indirectly quantify \gls{stp} attachment. Prior to the assay, the supernatent was filtered through a \SI{40}{\um} cell strainer to remove any stray microcarriers, which could increase the \gls{bca} readout as the assay is protein-agnostic and each microcarrier is approximately \SI{1}{\ug}. The carriers were washed analogously to the previous wash step to remove biotin, except two wash cycles were used, the agitation time was \SI{30}{\minute}, and the first cycle had an extra \SI{400}{\ul} \gls{pbs} to make up for the volume removed for the \gls{bca} assay. To coat with \glspl{mab}, sterile \product{\gls{bsa}}{\sigald}{A9576} was first added to a final concentration of \SI{2}{\percent} in order to prevent non-specific binding of the \glspl{mab} to the reaction tubes. Biotinylated \glspl{mab} against human CD3 \catnum{\bl}{317320} and CD28 \catnum{\bl}{302904} were combined in a 1:1 mass ratio and added to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\carrier}}. \glspl{mab} were allowed to bind to the carriers for \SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, \SI{400}{\ul} supernatant was sampled to indirectly quantify \gls{mab} attachment using an \product{\anti{\gls{igg}} \gls{elisa} kit}{Abcam}{157719}. Fully functionalized \glspl{dms} were washed in sterile \gls{pbs} analogous to the previous washing step to remove excess \gls{stp}. \begin{table}[!ht] \centering \caption{Microcarrier properties} \label{tab:carrier_props} \input{../tables/carrier_properties.tex} \end{table} Finished \glspl{dms} were washed once again in the cell culture media (analogous to previous washing steps) to be used for the T cell expansion. The concentration of the final \gls{dms} suspension was found by taking a \SI{50}{\uL} sample, plating in a well, and imaging the entire well. The image was then manually counted to obtain a concentration. Surface area for \si{\ab\per\um\squared} was calculated using the properties for \gls{cus} and \gls{cug} as given by the manufacturer \cref{tab:carrier_props}. \subsection{DMS Quality Control Assays} Biotin was quantified using the \product{\gls{haba} assay}{\sigald}{H2153-1VL}. In the case of quantifying \gls{snb} prior to adding it to the microcarriers, the sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar} NaOH and allowed to react for \SI{1}{\minute} in order to prevent the reactive ester linkages from binding to the avidin proteins in the \gls{haba}/avidin premix. All quantifications of \gls{haba} were performed on an Eppendorf D30 Spectrophotometer using \product{\SI{70}{\ul} cuvettes}{BrandTech}{759200}. The extinction coefficient at \SI{500}{\nm} for \gls{haba}/avidin was assumed to be \SI{34000}{\per\cm\per\molar}\footnote{\SI{500}{\nm} is normally used for the \gls{haba} assay, but the spectrophotometer to which we had access only had \SI{490}{\nm} as the closest wavelength; the extinction coefficient should change little}. The \gls{stp} binding to the microcarriers was quantified indirectly using a \product{\gls{bca} kit}{\thermo}{23227} according to the manufacturer’s instructions, with the exception that the standard curve was made with known concentrations of purified \gls{stp} instead of \gls{bsa}. Absorbance at \SI{592}{\nm} was quantified using a \pltread{}. The \gls{mab} binding to the microcarriers was quantified indirectly using an \gls{elisa} assay per the manufacturer’s instructions, with the exception that the same \glspl{mab} used to coat the carriers were used as the standard for the \gls{elisa} standard curve. This assay was quantified using a \pltread{}. Open biotin binding sites on the \glspl{dms} after \gls{stp} coating was quantified indirectly using \product{\gls{fitcbt}}{\thermo}{B10570}. Briefly, \SI{400}{\pmol\per\ml} \gls{fitcbt} were added to \gls{stp}-coated carriers and allowed to react for \SI{20}{\minute} at room temperature under constant agitation. The supernatant was quantified against a standard curve of \gls{fitcbt} using a \pltread{}. \Gls{stp} binding was verified after the \gls{stp}-binding step visually by adding \gls{fitcbt} to the \gls{stp}-coated \glspl{dms}, resuspending in \SI{1}{\percent} agarose gel, and imaging on a \product{lightsheet microscope}{Zeiss}{Z.1}. Overall \gls{mab} binding was verified visually by first staining with \product{\anti{\gls{igg}}-\gls{fitc}}{\bl}{406001}, incubating for \SI{30}{\minute}, washing with \gls{pbs}, and imaging on a confocal microscope. \subsection{T Cell Culture}\label{sec:tcellculture} Cryopreserved primary human T cells were either obtained as sorted \product{\cdp{3} T cells}{Astarte Biotech}{1017} or isolated from \product{\glspl{pbmc}}{Zenbio}{SER-PBMC} using a negative selection \product{\cdp{3} \gls{macs} kit}{\miltenyi}{130-096-535}. T cells were activated using \glspl{dms} or \product{\SI{3.5}{\um} CD3/CD28 magnetic beads}{\miltenyi}{130-091-441}. In the case of beads, T cells were activated at the manufacturer recommended cell:bead ratio of 2:1. In the case of \glspl{dms}, cells were activated using \SI{1500}{\dms\per\cm\squared} unless otherwise noted. Initial cell density was \SIrange{2e6}{2.5e6}{\cell\per\ml} to in a 96 well plate with \SI{300}{\ul} volume. Serum-free media was either \product{OpTmizer}{\thermo}{A1048501} or \product{TexMACS}{\miltenyi}{170-076-307} supplemented with \SIrange{100}{400}{\IU\per\ml} \product{recombinant human \il{2}}{Peprotech}{200-02} unless otherwise noted. Cell cultures were expanded for \SI{14}{\day} as counted from the time of initial seeding and activation. Cell counts and viability were assessed using \product{trypan blue}{\thermo}{T10282} or \product{\gls{aopi}}{Nexcelom Bioscience}{CS2-0106-5} and a \product{Countess Automated Cell Counter}{Thermo Fisher}{Countess 3 FL}. Media was added to cultures every \SIrange{2}{3}{\day} depending on media color or a \SI{300}{\mg\per\deci\liter} minimum glucose threshold. Media glucose was measured using a \product{GlucCell glucose meter}{Chemglass}{CLS-1322-02}. Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul} \product{\gls{stppe}}{\bl}{405204} and \SI{2}{ul} \product{\acd{45}-\gls{af647}}{\bl}{368538}, incubating for \SI{1}{\hour}, and imaging on a spinning disk confocal microscope. In the case of \gls{grex} bioreactors, we either used a \product{24 well plate}{Wilson Wolf}{P/N 80192M} or a \product{6 well plate}{Wilson Wolf}{P/N 80240M}. \subsection{Quantifying Cells on DMS Interior} To visualize T cells on the interior of the \glspl{dms}, we stained them with \gls{mtt}. \glspl{dms} with attached and loosely attached cells were sampled as desired and filtered through a \SI{40}{\um} cell strainer. While still in the cell strainer, \glspl{dms} were washed twice with \gls{pbs} and then dried by pulling liquid through the bottom of the cell strainer via a micropipette and dabbing with a KimWipe. \glspl{dms} were transferred to a 24 well plate with \SI{400}{\ul} media. \SI{40}{\ul} \gls{mtt} was added to each well and allowed to incubate for \SI{3}{\hour}, after which \glspl{dms} with cell were visualized via a brightfield microscope. To quantify cells on the interior of \glspl{dms}, cells and \glspl{dms} were isolated analogously to those for the \gls{mtt} stain up until the drying step. Cells were then transferred to a tube containing \SI{400}{\ul} at \SI{5}{\mg\per\ml} dispase solution. \glspl{dms} were incubated and rotated for \SI{45}{\minute} at \SI{37}{\degreeCelsius}, after which cells were counted as already described in \cref{sec:tcellculture}. \subsection{Quantification of Apoptosis Using Annexin-V} Apoptosis was quantified using \gls{anv} according to the manufacturer's instructions. Briefly, cells were transferred to flow tubes and washed twice with \gls{pbs} by adding \SI{3}{\ml} to each tube, centrifuging for \SI{400}{\gforce}, and aspirating the liquid down to \SI{200}{\ul}. The cells were analogously washed a third time with staining buffer (\SI{10}{\mM} \gls{hepes}, \SI{140}{\mM} NaCl, \SI{2.5}{\mM} \ce{CaCl2}) and aspirated down to a final volume of \SI{100}{\ul}. Cells were stained in this volume with \SI{1}{\ul} \product{\gls{anv}-\gls{fitc}}{\bl}{640906} and \SI{5}{\ul} \product{\gls{pi}}{\thermo}{P3566} and incubated for \SI{15}{\minute} at \gls{rt} in the dark. After incubation, \SI{400}{\ul} staining buffer was added to each tube. Each tube was then analyzed via flow cytometry. \subsection{Quantification of Caspase-3/7} \Gls{cas37} was quantified using \product{CellEvent dye}{\thermo}{C10723} according the manufacturer's instructions. Briefly, a 2X (\SI{8}{\mM}) working solution of CellEvent dye was added to \SI{100}{\ul} cell suspension (at least \num{5e4} cells) and incubated at \SI{37}{\degreeCelsius} for \SI{30}{\minute}. After incubation, cells were immediately analyzed via flow cytometry. \subsection{Quantification of BCL-2} \Gls{bcl2} was quantified using an \product{Human Total Bcl-2 DuoSet \gls{elisa} kit}{Rnd Systems}{DYC827B-2} according to the manufacturer's instructions and supplemented with \product{\gls{tmb} substrate solution}{eBioscience}{00-4201-56}, \product{5X diluent buffer}{\bl}{421203}, and \SI{2}{\normal} \ce{H2SO4} stop solution made in house. Briefly, cells were lysed using \product{10X lysis buffer}{Cell Signaling}{9803S}, and the lysate was quantified for protein using a \product{\gls{bca} assay}{\thermo}{23225} as directed. Standardized lysates were measured using the \gls{elisa} kit as directed. \subsection{Chemotaxis Assay} Migratory function was assayed using a transwell chemotaxis assay as previously described\cite{Hromas1997}. Briefly, \SI{3e5}{\cell} were loaded into a \product{transwell plate with \SI{5}{\um} pore size}{Corning}{3421} with the basolateral chamber loaded with \SI{600}{\ul} media and 0, 250, or \SI{1000}{\ng\per\mL} \product{CCL21}{Peprotech}{250-13}. The plate was incubated for \SI{4}{\hour} after loading, and the basolateral chamber of each transwell was quantified for total cells using \product{countbright beads}{\thermo}{C36950}. The final readout was normalized using the \SI{0}{\ng\per\mL} concentration as background. \subsection{Degranulation Assay} Cytotoxicity of expanded \gls{car} T cells was assessed using a degranulation assay as previously described\cite{Schmoldt1975}. Briefly, \num{3e5} T cells were incubated with \num{1.5e5} target cells consisting of either \product{K562 wild type cells}{ATCC}{CCL-243} or \cdp{19} K562 cells transformed with \gls{crispr} (kindly provided by Dr.\ Yvonne Chen, UCLA)\cite{Zah2016}. Cells were seeded in a flat bottom 96 well plate with \SI{1}{\ug\per\ml} \product{\acd{49d}}{eBioscience}{16-0499-81}, \SI{2}{\micro\molar} \product{monensin}{eBioscience}{ 00-4505-51}, and \SI{1}{\ug\per\ml} \product{\acd{28}}{eBioscience}{302914} (all functional grade \glspl{mab}) with \SI{250}{\ul} total volume. After \SI{4}{\hour} incubation at \SI{37}{\degreeCelsius}, cells were stained for CD3, CD4, and CD107a and analyzed on a \bd{} LSR Fortessa. Readout was calculated as the percent \cdp{107a} cells of the total \cdp{8} fraction. \subsection{CAR Expression} \gls{car} expression of the \anti{CD19} \gls{car} was quantified as previously described\cite{Zheng2012}. Briefly, cells were washed once and stained with \product{biotinylated \gls{ptnl}}{\thermo}{29997}. After a subsequent wash, cells were stained with \product{\gls{pe}-\gls{stp}}{\bl}{405204}, washed again, and analyzed on a \bd{} Accuri. Readout was percent \gls{pe}+ cells as compared to secondary controls (\gls{pe}-\gls{stp} with no \gls{ptnl}). \gls{car} expression of the \anti{\gls{bcma}} \gls{car} was quantified using a \product{\gls{fitc}-labeled \gls{bcma} protein}{Acro}{Bca-hf254}. \SI{100}{\ng} was added to tubes analogously to \gls{ptnl} and incubated for \SI{45}{\minute} prior to analyzing on a \bd{} Accuri \subsection{CAR Plasmid and Lentiviral Transduction}\label{sec:transduction} The anti-CD19-CD8-CD137-CD3$\upzeta$ \gls{car} sequence with the EF1$\upalpha$ promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a \product{FUGW transfer plasmid}{Addgene}{14883} kindly provided by the Emory Viral Vector Core. Lentiviral vectors were synthesized by the Emory Viral Vector Core or the Cincinnati Children's Hospital Medical Center Viral Vector Core. RNA titer was calculated using a \product{Lenti-X \gls{qpcr} titer kit}{Takara}{631235}. To transduce primary human T cells, \product{retronectin}{Takara}{T100A} was coated onto non-TC treated 96 well plates and used to immobilize lentiviral vector particles according to the manufacturer's instructions. Briefly, retronectin solution was adsorbed overnight at \SI{4}{\degreeCelsius} and blocked the next day using \gls{bsa}. Prior to transduction, lentiviral supernatant was spinoculated at \SI{2000}{\gforce} for \SI{2}{\hour} at \SI{4}{\degreeCelsius}. T cells were activated in 96 well plates using beads or \glspl{dms} for \SI{24}{\hour}, and then cells and beads/\glspl{dms} were transferred onto lentiviral vector coated plates and incubated for another \SI{24}{\hour}. Cells and beads/\glspl{dms} were removed from the retronectin plates using vigorous pipetting and transferred to another 96 well plate wherein expansion continued. % METHOD fill in missing product numbers \gls{bcma} \gls{car} lentiviral vector was synthesized in house as follows\footnote{lentiviral synthesis was performed by Ritika Jain in our laboratory and included here for reference}. \SI{10}{\ng} of \anti{\gls{bcma}}-CD8-CD137-CD3$\upzeta$ plasmid (generously provided by Jim Kochenderfer at the NIH)\cite{Lam2020} was added to \SI{50}{\ul} \product{DH5$\upalpha$ cells}{\thermo}{18265017} and incubated for \SI{30}{\minute} on ice. The cell mixture was then heat-shocked at \SI{42}{\degreeCelsius} for \SI{20}{\minute} before being placed on ice for another \SI{2}{\minute}. \SI{950}{\ul} luria broth was added to the cells which were then centrifuged for \SI{1}{\hour} at \SI{225}{\rpm}. \SI{20}{\ul} of the cell mixture was then spread over selection plates and incubated overnight at \SI{37}{\degreeCelsius}. Colonies were selected the following day and incubated in luria broth with \product{ampicillin}{\sigald{}}{A9518-5G} at \SI{37}{\degreeCelsius} for \SIrange{12}{16}{\hour} prior to using the \product{miniprep kit}{Qiagen}{27104} as per the manufacturer's instructions to isolate the plasmid DNA. Transfer plasmid along with \product{pMDLg/pRRE}{Addgene}{12251}, \product{pRSV-Rev}{Addgene}{12253}, and \product{pMD2.G}{Addgene}{12259} (generously provided by the Sloan lab at Emory University) in \product{Opti-Mem}{\thermo}{31-985-070} with \product{lipfectamine 2000}{\thermo}{11668019} were added dropwise to HEK 293T cells and incubated for \SI{6}{\hour}, after which all media was replaced with fresh fresh media. After \SI{24}{\hour} and \SI{48}{\hour}, supernatent was collected, pooled, and concentrated using a \product{Lenti-X concentrator}{Takara}{631231} prior to storing at \SI{-80}{\degreeCelsius}. \subsection{Sulfo-NHS-Biotin Hydrolysis Quantification} The equation for hydrolysis of \gls{snb} to biotin and \gls{nhs} is given by \cref{chem:snb_hydrolysis}. \begin{equation} \label{chem:snb_hydrolysis} \ce{NHS-CO-Biotin + OH- -> NHS- + Biotin-COOH} \end{equation} Measuring the hydrolysis of \gls{snb} was performed spectroscopically as the extinction coefficient of \ce{NHS-} is well-known. \gls{snb} was added to either \gls{di} water or \gls{pbs} in a UV-transparent 96 well plate. Kinetic analysis using a \pltread{} began immediately after prep, and readings at \SI{260}{\nm} were taken every minute for \SI{2}{\hour}. The extinction coefficient of \ce{NHS-} at \SI{260}{\nm} was assumed to be \SI{8600}{\per\cm\per\molar}. \subsection{Reaction Kinetics Quantification} The reaction kinetics of \gls{stp} attaching to biotin-coated microcarriers was determined experimentally. \SI{40}{\ug\per\ml} \gls{stp} was added to multiple batches of biotin-coated microcarriers, and supernatents were taken at fixed intervals and quantified for \gls{stp} protein using the \gls{bca} assay as described above. To model diffusion in the microcarriers, we assumed that its pores were large enough that the interactions between the \gls{stp} and surfaces would be small. This means that the apparent, macroscropic diffusion of a given species within the microcarriers would only depend on the aqueous diffusion coefficient of \gls{stp} and a fractional factor (the ``geometric diffusivity'') representing the additional path length an \gls{stp} molecule would take in the microcarriers due to the tortuousity and void fraction of its pore network. This is given in \cref{eqn:stp_diffusion_3}. \begin{equation} \label{eqn:stp_diffusion_3} \gls{sym:appdiff}=\gls{sym:diff} \gls{sym:geodiff} \end{equation} This geometric diffusivity of the microcarriers was determined using a pseudo-steady-state model. Each microcarrier was assumed to be a porous sphere with a fixed number of uniformly distributed ``receptors'' equal to the number of \gls{stp} molecules (here called ``ligands'') experimentally determined to bind to the microcarriers. Because the reaction rate between biotin and \gls{stp} is so fast (it is the strongest non-covalent bond in known existence), we assumed that the interface of unbound receptors (free biotin) shrunk as a function of \gls{stp} diffusing to the unbound biotin interface until the center of the microcarriers was reached. This model was given by \cref{eqn:stp_diffusion_1,eqn:stp_diffusion_2}: \begin{equation} \label{eqn:stp_diffusion_1} \frac{d\gls{sym:rad}}{d\gls{sym:time}} = \frac{- \gls{sym:appdiff} \gls{sym:bulkligconc}} {\gls{sym:rad} (1 - \gls{sym:rad} / \gls{sym:mcrad}) \evalat{\gls{sym:mcrecconc}}{\gls{sym:time} = 0}} \end{equation} \begin{equation} \label{eqn:stp_diffusion_2} \frac{d\gls{sym:bulkligconc}}{d\gls{sym:time}} = \frac{-4 \pi \gls{sym:mcnum} \gls{sym:appdiff}\gls{sym:bulkligconc}} {\gls{sym:vol} (1 / \gls{sym:rad} - 1 / \gls{sym:mcrad})} \end{equation} The diffusion rate of \gls{stp} was assumed to be \SI{6.2e-7}{\cm\squared\per\second}\cite{Kamholz2001}. Since all but $\beta$ was known, the experimental data was fit using these equations using \inlinecode{ode45} in MATLAB and least squares as the fitting error. These fitted equations were then used to simulate the reaction profile of \glspl{mab} assuming a diffusion rate of \SI{4.8e-7}{\cm\squared\per\second}\cite{Sherwood1992}. To model the washing of the microcarriers, they once again were assumed to be porous spheres filled with whatever amount of reagent was left unbound from the previous step (which was assumed to be equal to concentration in the supernatent). The fitted geometric diffusivity from above was used in these washing calculations, and \SI{5.0e-6}{\cm\squared\per\second}\cite{Niether2020} was used as the diffusion coefficient for free biotin. The diffusion out of the microcarriers is given by the following partial differential equation and boundary conditions: \begin{equation} \label{eqn:stp_washing} \frac{\partial \gls{sym:mcligconc}}{\partial \gls{sym:time}} = \frac{1}{\gls{sym:rad}^2} \frac{\partial}{\partial \gls{sym:rad}} \left(\gls{sym:rad}^2 \gls{sym:appdiff} \frac{\partial \gls{sym:mcligconc}}{\partial \gls{sym:rad}} \right) \end{equation} \begin{equation} \label{eqn:stp_washing_time_bc} \evalat{\gls{sym:mcligconc}}{\gls{sym:time}=0} = \evalat{\gls{sym:bulkligconc}}{\gls{sym:time}=0} \end{equation} \begin{equation} \label{eqn:stp_washing_left_bc} \gls{sym:mcflux}\rvert_{\gls{sym:rad}=0} = 0 \end{equation} \begin{equation} \label{eqn:stp_washing_right_bc} \evalat{\gls{sym:mcligconc}}{\gls{sym:rad} = \gls{sym:mcrad}} = (\evalat{\gls{sym:bulkligconc}}{\gls{sym:time} = 0} + \evalat{\gls{sym:bulkligconc}}{\gls{sym:time} = \infty}) / 2 \end{equation} In order to avoid solving a moving boundary value problem, the concentration at the boundary of the microcarriers was fixed at the average of the final and initial concentration expected to be observed in bulk. This should be a reasonable assumption given that the volume inside the microcarriers is tiny compared to the amount of volume added in the wash, thus the boundary concentration should change little. All diffusion coefficients were taken to be valid at \gls{rt} and in \gls{di} water, which is a safe assumption given that our reaction medium was 1X \gls{pbs}. See \cref{sec:appendix_binding} and \cref{sec:appendix_washing} for the MATLAB code and derivations, as well as output in the case of the washing steps. \subsection{Luminex Analysis}\label{sec:luminex_analysis} Luminex was performed using a \product{ProcartaPlex kit}{\thermo}{custom} for the markers outlined in \cref{tab:luminex_panel} with modifications (note that some markers were run in separate panels to allow for proper dilutions). Briefly, media supernatents from cells were sampled as desired and immediately placed in a \SI{-80}{\degreeCelsius} freezer until use. Before use, samples were thawed at \gls{rt} and vortexed to ensure homogeneity. To run the plate, \SI{25}{\ul} of magnetic beads were added to the plate and washed 3X using \SI{300}{\ul} of wash buffer. \SI{25}{\ul} of samples or standard were added to the plate and incubated for \SI{120}{\minute} at \SI{850}{\rpm} at \gls{rt} before washing analogously 3X with wash buffer. \SI{12.5}{\ul} detection \glspl{mab} and \SI{25}{\ul} \gls{stppe} were sequentially added, incubated for \SI{30}{\minute} and vortexed, and washed analogously to the sample step. Finally, samples were resuspended in \SI{120}{\ul} reading buffer and analyzed via a BioRad Bioplex 200 plate reader. An 8 point log\textsubscript{2} standard curve was used, and all samples were run with single replicates. Luminex data was preprocessed using R for inclusion in downstream analysis as follows. Any cytokine level that was over-range (`OOR >' in output spreadsheet) was set to the maximum value of the standard curve for that cytokine. Any value that was under-range (`OOR <' in output spreadsheet) was set to zero. All values that were extrapolated from the standard curve were left unchanged. \begin{table}[!ht] \centering \caption{Luminex panel} \label{tab:luminex_panel} \input{../tables/luminex_panel.tex} \end{table} \subsection{Data Aggregation and Meta-Analysis} In order to perform meta-analysis on all experimental data generate using the \gls{dms} system, we developed a program to curate and aggregate the raw datafiles into a \gls{sql} database (\cref{sec:appendix_meta}). The data files to be aggregated included Microsoft Excel files which held timeseries measurements for cell cultures (eg cell counts, viability, glucose, \il{2} added, media added, and media removed), \gls{fcs} files for cellular phenotypes, and FlowJo files which held gating parameters and statistics based on the \gls{fcs} files. Additional information which was held in electronic lab notebooks (eg OneNote files) was not easily parsable, and thus this data was summarized in YAML files. The data included in these YAML files included reagent characteristics (vendor, catalog number, lot number, manufacturing date), cell donor characteristics (age, \gls{bmi}, phenotype, demographic, gender), and all experimental parameters such as the number of beads or \glspl{dms} added. To aggregate the data in a database, we wrote a program using Python, R, and Docker which retrieved the data from its source location and inserted the data in a PostgreSQL database (specifically the Aurora implementation hosted on \gls{aws}). This program included checks to ensure the integrity of source data (for example, flagging entries which had a reagent whose manufacturing date was after the date the experiment started, which signifies a human input error). \subsection{Statistical Analysis}\label{sec:statistics} For 1-way \gls{anova} analysis with Tukey multiple comparisons test, significance was assessed using the \inlinecode{stat\_compare\_means} function with the \inlinecode{t.test} method from the \inlinecode{ggpubr} library in R. For 2-way \gls{anova} analysis, significance of main and interaction effects was determined using the \inlinecode{car} library in R. For least-squares linear regression, statistical significance was evaluated the \inlinecode{lm} function in R. All results with categorical variables are reported relative to baseline reference. Each linear regression was assessed for validity using residual plots (to assess constant variance and independence assumptions), QQplots and Shapiro-Wilk normality test (to assess normality assumptions), Box-Cox plots (to assess need for power transformations), and lack-of-fit tests where replicates were present (to assess model fit in the context of pure error). Significance was evaluated at $\upalpha$ = 0.05. \subsection{Flow Cytometry}\label{sec:flow_cytometry} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/gating_strategy.png} \endgroup \caption[Gating Strategy] {Gating strategy for quantifying \ptmemp{}, \pthp{}, and \ptcarp{}.} \label{fig:gating_strategy} \end{figure*} \begin{table}[!ht] \centering \caption{Antibodies used for flow cytometry} \label{tab:flow_mabs} \input{../tables/flow_mabs.tex} \end{table} All \glspl{mab} used for flow cytometry are shown in \cref{tab:flow_mabs}. Other reagents for specialized assays such as degranulation are described in their respective sections. Cells were gated according to \cref{fig:gating_strategy}. \section{Results} \subsection{DMSs Can be Fabricated in a Controlled Manner} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/dms_coating.png} \phantomsubcaption\label{fig:cug_vs_cus} \phantomsubcaption\label{fig:biotin_coating} \phantomsubcaption\label{fig:stp_coating} \phantomsubcaption\label{fig:mab_coating} \phantomsubcaption\label{fig:stp_carrier_fitc} \phantomsubcaption\label{fig:mab_carrier_fitc} \endgroup \caption[\acrshort{dms} Coating] {\gls{dms} functionalization results. \subcap{fig:cug_vs_cus}{Bound \gls{stp} surface density on either \gls{cus} or \gls{cug} microcarriers. Surface density was estimated using the properties in~\cref{tab:carrier_props}}. Total binding curve of \subcap{fig:biotin_coating}{biotin}, \subcap{fig:stp_coating}{\gls{stp}}, and \subcap{fig:mab_coating}{\glspl{mab}} as a function of biotin added for batches manufactured on different dates. \subcap{fig:stp_carrier_fitc}{\gls{stp}-coated or uncoated \glspl{dms} treated with \gls{fitcbt} and imaged using a lightsheet microscope.} \subcap{fig:mab_carrier_fitc}{\gls{mab}-coated or \gls{stp}-coated \glspl{dms} treated with \anti{\gls{igg}} \glspl{mab} and imaged using a lightsheet microscope.} } \label{fig:dms_coating} \end{figure*} Two types of gelatin-based microcariers, \gls{cus} and \gls{cug}, were covalently conjugated with varying concentration of \gls{snb} and then coated with \gls{stp} and \glspl{mab} to make \glspl{dms}. Aside from slight differences in swelling ratio and crosslinking chemistry\cite{purcellmain} the properties of \gls{cus} and \gls{cug} were the same (\cref{tab:carrier_props}). We chose to continue with the \gls{cus}-based \glspl{dms}, which showed higher overall \gls{stp} binding compared to \gls{cug}-based \glspl{dms} (\cref{fig:cug_vs_cus}). We showed that by varying the concentration of \gls{snb}, we were able to control the amount of attached biotin (\cref{fig:biotin_coating}), mass of attached \gls{stp} (\cref{fig:stp_coating}), and mass of attached \glspl{mab} (\cref{fig:mab_coating}). Furthermore, we showed that the microcarriers were evenly coated with \gls{stp} on the surface and throughout the interior as evidenced by the presence of biotin-binding sites occupied with \gls{fitcbt} on the microcarrier surfaces after the \gls{stp}-coating step (\cref{fig:stp_carrier_fitc}). Finally, we confirmed that biotinylated \glspl{mab} were bound to the \glspl{dms} by staining either \gls{stp}- or \gls{stp}/\gls{mab}-coated carriers with \antim{\gls{igg}-\gls{fitc}} and imaging on a confocal microscope (\cref{fig:mab_carrier_fitc}). Taking this together, we noted that the maximal \gls{mab} binding capacity occurred near \SI{50}{\nmol} biotin input (which corresponded to \SI{2.5}{\nmol\per\mg\of{\dms}}) thus we used this in subsequent processes. We then asked how sensitive the \gls{dms} manufacturing process was to a variety of variables. In particular, we focused on the biotin-binding step, since it appeared that the \gls{mab} binding was quadratically related to biotin binding (\cref{fig:mab_coating}) and thus controlling the biotin binding step would be critical to controlling the amount and \glspl{mab} and thus the amount of signal the T cells receive downstream. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/dms_qc.png} \phantomsubcaption\label{fig:dms_qc_doe} \phantomsubcaption\label{fig:dms_qc_ph} \phantomsubcaption\label{fig:dms_qc_washes} \phantomsubcaption\label{fig:dms_snb_decay_curves} \endgroup \caption[\acrshort{dms} Process Parameters] {Investigation of influential parameters for the \gls{dms} process. \subcap{fig:dms_qc_doe}{\gls{doe} investigating the effect of initial mass of microcarriers, reaction temperature, and biotin concentration on biotin attachment.} \subcap{fig:dms_qc_ph}{Effect of reaction ph on biotin attachment.} \subcap{fig:dms_qc_washes}{effect of post-autoclave washing of the microcarriers on biotin attachment.} \subcap{fig:dms_snb_decay_curves}{Hydrolysis curves of \gls{snb} in \gls{pbs} or \gls{di} water.} All statistical tests where p-values are noted are given by two-tailed t tests. } \label{fig:dms_qc} \end{figure*} To answer this question, we first performed a \gls{doe} to understand the effect of reaction parameters on biotin binding. The parameters included in this \gls{doe} were temperature, microcarrier mass, and \gls{snb} input mass. These were parameters that we specifically controlled but hypothesized might have some sensitivity on the final biotin mass attachment rate depending on their noise and uncertainty. In particular, temperature was ``controlled'' only by allowing the microcarrier suspension to come to \gls{rt} after autoclaving. After performing a full factorial \gls{doe} with three center points as the target reaction conditions, we found that the final biotin binding mass is only highly dependent on biotin input concentration (\cref{fig:dms_qc_doe}). Overall, temperature had no effect and carrier mass had no effect at higher temperatures, but carrier mass had a slightly positive effect when temperature was low. This could be because lower temperature might make spontaneous decay of \gls{snb} occur slower, which would give \gls{snb} molecules more opportunity to diffuse into the microcarriers and react with amine groups to form attachments. It seemed that concentration only has a linear effect and doesn't interact with any of the other variables, which is not surprising given the behavior observed in (\cref{fig:biotin_coating}) We also observed that the reaction pH does not influence the amount of biotin attached (\cref{fig:dms_qc_ph}), which indicates that while higher pH might increase the number of deprotonated amines on the surface of the microcarrier, it also increases the number of \ce{OH-} groups which can spontaneously hydrolyze the \gls{snb} in solution (\cref{chem:snb_hydrolysis}). Furthermore, we observed that washing the microcarriers after autoclaving increased the biotin binding rate (\cref{fig:dms_qc_washes}). While we did not explore this further, one possible explanation for this behavior is that the microcarriers have some loose protein in the form of powder or soluble peptides that competes for \gls{snb} binding against the surface of the microcarriers, and when measuring the supernatent using the \gls{haba} assay, these soluble or lightly-suspended peptides/protein fragments are also measured and therefore inflate the readout. Lastly, we asked what the effect on reaction pH had on spontaneous degradation of \gls{snb} while in solution (\cref{chem:snb_hydrolysis}). If the \gls{snb} significantly degrades within minutes of preparation, then it is important to carefully control the timing between \gls{snb} solution preparation and addition to the microcarriers. We found that in the presence of \gls{di} water, \gls{snb} is extremely stable (\cref{fig:dms_snb_decay_curves}) where it decays rapidly in the presence of \gls{pbs} buffered to pH of 7.1. In fact, the \gls{di} water curve actually decreased slightly, possibly due to \gls{snb} absorbing to the plate surface. \gls{snb} is known to hydrolyze in the presence of \ce{OH-} groups, but the lack of hydrolysis in \gls{di} water can be explained by the fact that biotin itself is acidic, and thus the reaction is self-inhibitory in an unbuffered and neutral pH system. Because we dissolve our \gls{snb} in \gls{di} water prior to adding it to the microcarrier suspension (which itself is in \gls{pbs}) this result indicated that hydrolysis is not of concern when adding \gls{snb} within minutes. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/dms_timing.png} \phantomsubcaption\label{fig:dms_biotin_rxn_mass} \phantomsubcaption\label{fig:dms_biotin_rxn_frac} \phantomsubcaption\label{fig:dms_stp_per_time} \phantomsubcaption\label{fig:dms_mab_per_time} \phantomsubcaption\label{fig:dms_biotin_washed} \endgroup \caption[\acrshort{dms} Reaction Kinetics] {Reaction kinetics for microcarrier functionalization. \subcap{fig:dms_biotin_rxn_mass}{Biotin mass bound per time} \subcap{fig:dms_biotin_rxn_frac}{Fraction of input biotin bound per time} \subcap{fig:dms_stp_per_time}{\Gls{stp} bound per time. Each dot is an experimental run and the line is the fitted model.} \subcap{fig:dms_mab_per_time}{Simulated \glspl{mab} bound per time.} \subcap{fig:dms_biotin_washed}{Biotin quantification via the \gls{haba} assay after washing.} } \label{fig:dms_kinetics} \end{figure*} \subsection{DMS Process Has Defined Reaction Kinetics} We investigated the reaction kinetics of all three coating steps (accompanying MATLAB codes are provided in \cref{sec:appendix_binding}). To quantify the reaction kinetics of the biotin binding step, we reacted multiple batches of \SI{20}{\mg\per\ml} microcarriers in \gls{pbs} at \gls{rt} with \gls{snb} in parallel and sacrificially analyzed each at varying timepoints using the \gls{haba} assay. This was performed at two different concentrations. We observed that for either concentration, the reaction was over in \SIrange{20}{30}{\minute} (\cref{fig:dms_biotin_rxn_mass}). Furthermore, when put in terms of fraction of input \gls{snb}, we observed that the curves are almost identical (\cref{fig:dms_biotin_rxn_frac}). Given this, the reaction step for biotin attached can be set to \SI{30}{\minute}\footnote{we actually used \SI{60}{\minute} as outlined in methods, which shouldn't make any difference except for costing more time}. Next, we quantified the amount of \gls{stp} reacted with the surface of the biotin-coated microcarriers. Different batches of biotin-coated \glspl{dms} were coated with \SI{40}{\ug\per\ml} \gls{stp} and sampled at intermediate timepoints using the \gls{bca} assay to indirectly quantify the amount of attached \gls{stp} mass. We found this reaction took approximately \SI{30}{\minute} (\cref{fig:dms_stp_per_time}). Assuming a quasi-steady-state paradigm, we used this experimental binding data to compute the geometric diffusivity of the microcarriers and fit a continuous model for the \gls{stp} binding reaction. We computed the number of ``receptors'' using the maximum mass observed to bind to the \gls{dms}, which should describe the upper-bound for reaction time (\cref{fig:stp_coating}). Using the diffusion rate of the \gls{stp} (\SI{6.2e-7}{\cm\squared\per\second}), we then calculated the geometric diffusivity of the microcarriers to be 0.190 (see \cref{eqn:stp_diffusion_1,eqn:stp_diffusion_2}). Using this geometric diffusivity and the known diffusion coefficient of \glspl{mab} in water, we calculated the binding of \glspl{mab} per time onto the microcarriers (this obviously assumes that the effectively diffusivity is independent of the protein used, which should be reasonable given that the pores of the microcarriers are huge compared to the proteins, and we don't expect any significant reaction between the protein and the microcarrier surface save for the \gls{stp}-biotin binding reaction). Once again, we used the maximum number of \glspl{mab} observed to determine the number of receptors for \glspl{mab} on the microcarriers, which should correspond to the upper-bound for the reaction time (\cref{fig:mab_coating}). According to this model, the \gls{mab} binding reaction should be complete within \SI{75}{\minute} under the conditions used for our protocol (\cref{fig:dms_mab_per_time})\footnote{we actually used \SI{60}{\minute} as describe in the method section as this model was not updated with new parameters until recently; however, we should point out that even at \SI{60}{\minute} the reaction appears to be >\SI{95}{\percent} complete}. Finally, we calculated the number of wash steps needed to remove the reagents between each step, including the time for each wash which required the geometric diffusivity of the microcarriers as calculated above. This is important, as failing to wash out residual free \gls{snb} (for example) could occupy binding sites on the \gls{stp} molecules, lowering the effective binding capacity of the \glspl{mab} downstream. Each wash was a 1:15 dilution (\SI{1}{\ml} reaction volume in a \SI{15}{\ml} conical tube), and in the case of \gls{snb} we wished to wash out enough biotin such that less than \SI{1}{\percent} of the binding sites in \gls{stp} would be occupied. Given this dilution factor, a maximum of \SI{20}{\nmol} of biotin remaining \cref{fig:biotin_coating} \SI{2.9}{\nmol} biotin binding sites on \SI{40}{\ug} \gls{stp} (assuming 4 binding sites per \gls{stp} protein), this turned out to be 3 washes. By similar logic, using 2 washes after the \gls{stp} binding step will ensure that the number of free \gls{stp} binding sites is less than 20X the number of \gls{mab} molecules added\footnote{This step may benefit from an additional wash, as the number of washes used here was determined when \SI{40}{\ug} rather than \SI{4}{\ug} \gls{mab} was used to coat the \gls{dms}, yielding a much wider margin. However, it is also not clear to what extent this matters, as the \gls{mab} have multiple biotin molecules per \gls{mab} protein, and thus one \gls{mab} would require binding to several \gls{stp} molecules to be prevented from binding at all.} To determine the length of time required for each wash, we again assumed the microcarriers to be porous spheres, this time with an initial concentration of \gls{snb}, \gls{stp}, or \glspl{mab} equal to the final concentration of the bulk concentration of the previous binding step, and calculated the amount of time it would take for the concentration profile inside the microcarriers to equilibrate to the bulk in the wash step. Using this model, we found that the wash time for \gls{snb}, \gls{stp}, and \glspl{mab} was \SI{3}{\minute}, \SI{15}{\minute}, and \SI{17}{\minute} respectively. We verified that the \gls{snb} was totally undetectable after washing (\cref{fig:dms_biotin_washed}). The other two species need to be verified in a similar manner; however, we should not that the washing time for both the \gls{stp} and \gls{mab} coating steps were \SI{30}{\minute}, which is a significant margin of safety (albeit one that could be optimized). MATLAB code and output for all wash step calculations are given in \cref{sec:appendix_washing}. \subsection{DMSs Can Efficiently Expand T Cells Compared to Beads} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/cells_on_dms.png} \phantomsubcaption\label{fig:dms_cells_phase} \phantomsubcaption\label{fig:dms_cells_fluor} \endgroup \caption[T Cells Growing on \acrshortpl{dms}] {Cells grow in tight clusters in and around functionalized \gls{dms}. \subcap{fig:dms_cells_phase}{Phase-contrast image of T cells growing on \glspl{dms}} \subcap{fig:dms_cells_fluor}{Confocal images of T cells in varying z-planes growing on \glspl{dms} on day 9. \Glspl{dms} were stained using \gls{stppe} (red) and T cells were stained using \acd{45}-\gls{af647}.} Images are from day 7 of culture. } \label{fig:dms_cells} \end{figure*} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/dms_expansion.png} \phantomsubcaption\label{fig:dms_expansion_bead} \phantomsubcaption\label{fig:dms_expansion_isotype} \endgroup \caption[\acrshortpl{dms} Selectively Expand T Cells] {T cells are selectively expanded on \gls{dms}. \subcap{fig:dms_expansion_bead}{T cells expanded with either \glspl{dms} or bead for 12 days. Significance was assessed using a two-tailed heteroschodastic T test.} \subcap{fig:dms_expansion_isotype}{T cells grown on \glspl{dms} coated with either activating \glspl{mab} or \gls{igg} isotype control \glspl{mab}.} } \label{fig:dms_expansion} \end{figure*} We next sought to determine how our \glspl{dms} could expand T cells compared to state-of-the-art methods used in industry. All bead expansions were performed as per the manufacturer’s protocol, with the exception that the starting cell densities were matched between the beads and \glspl{dms} to ~\SI{2.5e6}{\cell\per\ml}. We observed that T cells in \gls{dms} culture grew in tight clumps on the surface of the \glspl{dms} as well as inside the pores of the \glspl{dms} (\cref{fig:dms_cells_phase,fig:dms_cells_fluor}). Furthermore, we observed that the \glspl{dms} conferred greater expansion compared to traditional beads, and significantly greater expansion after \SI{12}{\day} of culture (\cref{fig:dms_expansion_bead}). We also observed no T cell expansion using \glspl{dms} coated with an isotype control mAb compared to \glspl{dms} coated with \acd{3}/\acd{28} \glspl{mab} (\cref{fig:dms_expansion_isotype}), confirming specificity of the expansion method. Given that \il{2} does not lead to expansion on its own, we know that the expansion of the T cells shown here is due to the \acd{3} and \acd{28} \glspl{mab}\cite{Waysbort2013}. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/apoptosis.png} \phantomsubcaption\label{fig:apoptosis_annV} \phantomsubcaption\label{fig:apoptosis_cas} \phantomsubcaption\label{fig:apoptosis_bcl2} \endgroup \caption[Apoptosis Quantification for \acrshortpl{dms}] {\glspl{dms} produce cells with lower apoptosis marker expression on average compared to bead. \subcap{fig:apoptosis_annV}{Quantification of apoptosis and necrosis by \gls{anv} and \gls{pi}.} \subcap{fig:apoptosis_cas}{Quantification of Caspase-3/7 expression using CellEvent dye.} \subcap{fig:apoptosis_bcl2}{Quantification of BCL-2 expression using \gls{elisa}. All statistical tests shown are two-tailed homoschodastic t-tests. All cells were harvested at day 8.} } \label{fig:dms_apoptosis} \end{figure*} Given that the \gls{dms} system seemed to expand T cells more effectively, we asked if this difference was due to a reduction in apoptosis or an increase in proliferation rate (or both). We assessed the apoptotic state of T cells grown using either bead or \gls{dms} harvested on day 8 using \gls{pi} and \gls{anv}. \gls{anv} is a marker which stains phospholipid phosphatidylserine, which is usually present only on the cytoplasmic surface of the cell membrane, but flips to the outside when the cell becomes apoptotic. \gls{pi} stains the nucleus of the cell, but only penetrates necrotic cells which have a perforated cell membrane. When staining for these two markers and assessing via flow cytometry, we observed that the \gls{dms}-expanded T cells have lower frequencies of apoptotic and necrotic cells (\cref{fig:apoptosis_annV}). Furthermore, we stained our cultures with CellEvent dye, an indicator of \gls{cas37} which is activated in apoptotic cells. In line with the \gls{pi}/\gls{anv} results, we observed that the \gls{dms} T cells had lower frequency of \gls{cas37} expression, indicating less apoptosis for our method (\cref{fig:apoptosis_cas}). Finally, we lysed our cells and stained for \gls{bcl2}, which is also upregulated in apoptosis. In this case, some (but not all) of the bead-expanded cultures showed higher \gls{bcl2} expression, which could indicate more apoptosis in those groups (\cref{fig:apoptosis_bcl2}). None of the \gls{dms} cultures showed similar heightened expression. Taken together, these data suggest that the \gls{dms} platform at least in part achieves higher expansion by lowering apoptosis. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/dms_inside.png} \phantomsubcaption\label{fig:dms_inside_bf} \phantomsubcaption\label{fig:dms_inside_regression} \endgroup \caption[T Cells Growing on Interior of \acrshortpl{dms}] {A percentage of T cells grow in the interior of \glspl{dms}. \subcap{fig:dms_inside_bf}{T cells stained dark with \gls{mtt} after growing on either coated or uncoated \glspl{dms} for 15 days visualized with brightfield microscope.} \subcap{fig:dms_inside_regression}{Linear regression performed on T cell percentages harvested on the interior of the \glspl{dms} vs the initial starting cell density.} } \label{fig:dms_inside} \end{figure*} \begin{table}[!ht] \centering \caption{Regression for fraction of cells in \acrshortpl{dms} at day 14} \label{tab:inside_regression} \input{../tables/inside_fraction_regression.tex} \end{table} We also asked how many cells were inside the \glspl{dms} instead of free-floating in suspension and/or loosely attached to the surface. We qualitatively verified the presence of cells inside the \glspl{dms} using a \gls{mtt} stain to opaquely mark cells and enable visualization on a brightfield microscope (\cref{fig:dms_inside_bf}). After seeding \glspl{dms} at different densities and expanding for \SI{15}{\day}, we filtered the \glspl{dms} out of the cell suspension and digested them using dispase to free any cells attached on the inner surface. We observed that approximately \SI{15}{\percent} of the total cells after \SI{15}{\day} were on the interior surface of the \glspl{dms} (\cref{fig:dms_inside_regression,tab:inside_regression}). Performing linear regression on this data revealed that the percentage of T cells inside the \glspl{dms} does not depend on the initial starting cell density (at least when harvested after \SI{15}{\day}) (\cref{tab:inside_regression}). \subsection{DMSs Lead to Greater Expansion and High-Quality Phenotypes} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/dms_vs_bead_expansion.png} \phantomsubcaption\label{fig:dms_exp_fold_change} \phantomsubcaption\label{fig:dms_exp_mem} \phantomsubcaption\label{fig:dms_exp_cd4} \phantomsubcaption\label{fig:dms_exp_mem4} \phantomsubcaption\label{fig:dms_exp_mem8} \endgroup \caption[\acrshort{dms} vs Bead Expansion] {\gls{dms} lead to superior expansion of T cells compared to beads across multiple donors. \subcap{fig:dms_exp_fold_change}{Longitudinal fold change of T cells grown using either \glspl{dms} or beads. Significance was evaulated using t tests at each timepoint} Fold change of subpopulations expanded using either \gls{dms} or beads at day 14, including \subcap{fig:dms_exp_mem}{\ptmem{} cells}, \subcap{fig:dms_exp_cd4}{\pth{} cells}, \subcap{fig:dms_exp_mem4}{\ptmemh{} cells}, and \subcap{fig:dms_exp_mem8}{\ptmemk{} cells}. \sigkey{} } \label{fig:dms_exp} \end{figure*} After observing differences in expansion, we further hypothesized that the \gls{dms} cultures could lead to a different T cell phenotype. In particular, we were interested in the formation of \glspl{tn}, \gls{tscm}, and \glspl{tcm} as these represent a subset with higher capacity to replicate and therefore improved clinical prognosis\cite{Gattinoni2011, Wang2018}. We measured the frequency of these subtypes by staining for CCR7 and CD62L. Using three donor lots, we noted again \glspl{dms} produced more T cells over a \SI{14}{\day} expansion than beads, with significant differences in number appearing as early as \SI{5}{\day} (\cref{fig:dms_exp_fold_change}). Furthermore, we noted that \glspl{dms} produced more memory/naïve cells after \SI{14}{\day} when compared to beads for all donors (\cref{fig:dms_exp_mem,fig:dms_exp_cd4}) showing that the \gls{dms} platform is able to selectively expand potent, early differentiation T cells. Of additional interest was the preservation of the CD4+ compartment. In healthy donor samples (such as those used here), the typical CD4:CD8 ratio is 2:1. We noted that \glspl{dms} produced more CD4+ T cells than bead cultures as well as naïve/memory, showing that the \gls{dms} platform can selectively expand CD4 T cells to a greater degree than beads \cref{fig:dms_exp_cd4}. The trends held true when observing the CD4+ and CD8+ fractions of the naïve/memory subset (\ptmem{}) (\cref{fig:dms_exp_mem4,fig:dms_exp_mem8}). \begin{figure*}[ht!] \begingroup \includegraphics{../figures/dms_phenotypes.png} \phantomsubcaption\label{fig:dms_phenotype_mem} \phantomsubcaption\label{fig:dms_phenotype_cd4} \endgroup \caption[Representative Flow Plots of \ptmem{} and \pth{} T Cells] {Representative flow plots of \ptmem{} and \pth{} T cells at day 14 of expansion using either beads or \glspl{dms}. For three representative donor samples, phenotypes are shown for \subcap{fig:dms_phenotype_mem}{\ptmem{}} and \subcap{fig:dms_phenotype_cd4}{\pth}. Each population was also gated on \cdp{3} T cells. } \label{fig:dms_phenotype} \end{figure*} We also observed that, at least among some donors and conditions\footnote{these results were not always consistent, see the metaanalysis at the end of this aim for an in-depth quantification of this observation} that the fraction of \ptmem{} and \pth{} T cells was higher in the \gls{dms} groups compared to the bead groups (\cref{fig:dms_phenotype})\footnote{these where not the same donors as used for \cref{fig:dms_exp}}. Note that in the case of \pthp{}, the donors we used had an initial \pthp{} that was much higher (healthy donors generally have a CD4:CD8 ratio of 2:1), so the proper interpretation of this is that the \pthp{} decreases less over the culture period with the \gls{dms} platform as opposed to the beads (or alternatively, the \gls{dms} has less preferential expansion for \cdp{8} T cells). We cannot say the same about the \ptmemp{} since we did not have the initial data for this phenotype; (although memory and naive cells should be the vast majority of cells given that \glspl{pbmc} is taken from blood which has mostly these cell types). Taken together, these data indicate the \gls{dms} platform has the capacity to expand higher numbers and percentages of highly potent \ptmem{} and \pth{} T cells compared to state-of-the-art bead technology. \subsection{DMSs Produce Functional CAR T Cells} After optimizing for naïve/memory and CD4 yield, we sought to determine if the \glspl{dms} were compatible with lentiviral transduction protocols used to generate \gls{car} T cells\cite{Tumaini2013, Lamers2014}. We added a \SI{24}{\hour} transduction step on day 1 of the \SI{14}{\day} expansion to insert an anti-CD19 \gls{car}\cite{Milone2009} with a \gls{moi} of 10 and subsequently measured the surface expression of the \gls{car} on day 14 (\cref{fig:car_cd19_flow,fig:car_cd19_endpoint}). We noted that there was robust \gls{car} expression in over \SI{25}{\percent} of expanded T cells, and there was no observable difference in \gls{car} expression between beads and \glspl{dms}. We also verified the functionality of expanded \gls{car} T cells using a degranulation assay\cite{Zheng2012}. Briefly, T cells were cocultured with target cells (either wild-type K562 or CD19-expressing K562 cells) for \SI{4}{\hour}, after which the culture was analyzed via flow cytometry for the appearance of CD107a on CD8+ T cells. CD107a is found on the inner-surface of cytotoxic granules and will emerge on the surface after cytotoxic T cells are activated and degranulate. Indeed, we observed degranulation in T cells expanded with both beads and \glspl{dms}, although not to an observably different degree (\cref{fig:car_degran_flow,fig:car_degran_endpoint}). Taken together, these results indicated that the \glspl{dms} provide similar transduction efficiency compared to beads. We also verified that expanded T cells were migratory using a chemotaxis assay for CCL21; since \glspl{dms} produced a larger percentage of naïve and memory T cells (which have CCR7, the receptor for CCL21) we would expect higher migration in \gls{dms}-expanded cells vs.\ their bead counterparts. Indeed, we noted a significantly higher migration percentage for T cells grown using \glspl{dms} versus beads (\cref{fig:car_degran_migration}). Interestingly, there also appeared to be a decrease in CCL21 migration between transduced and untransduced T cells expanded using beads, but this interaction effect was only weakly significant (p = 0.068). No such effect was seen for \gls{dms}-expanded T cells, showing that migration was likely independent of \gls{car} transduction. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/car_cd19.png} \phantomsubcaption\label{fig:car_cd19_flow} \phantomsubcaption\label{fig:car_cd19_endpoint} \endgroup \caption[CD19 Transduction] {\glspl{dms} lead to efficient CD19 transduction. \subcap{fig:car_cd19_flow}{Representative flow cytometry plot for transduced or untransduced T cells stained with \gls{ptnl}.} \subcap{fig:car_cd19_endpoint}{Endpoint plots with \gls{anova} for transduced or untransduced T cells stained with \gls{ptnl}.} All data is from T cells expanded for \SI{14}{\day}. } \label{fig:car_cd19} \end{figure*} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/car_degranulation.png} \phantomsubcaption\label{fig:car_degran_flow} \phantomsubcaption\label{fig:car_degran_endpoint} \phantomsubcaption\label{fig:car_degran_migration} \endgroup \caption[\acrshort{car} T Cell Functionality] {\glspl{dms} produce functional \gls{car} T cells. \subcap{fig:car_degran_flow}{Representative flow plot for degranulating T cells.} \subcap{fig:car_degran_endpoint}{Endpoint plots for transduced or untransduced T cells stained with \cd{107a} for the degranulation assay.} \subcap{fig:car_degran_migration}{Endpoint plot for transmigration assay with \gls{anova}.} All data is from T cells expanded for \SI{14}{\day}. } \label{fig:car_degran} \end{figure*} In addition to CD19 \gls{car} T cells, we also demonstrated that the \gls{dms} platform can be used to expand \gls{car} T cells against \gls{bcma}. Analogous to CD19, \gls{dms} and bead produced similar fractions of \ptcar{} cells (albeit in this case at day 7 and with an undefined \gls{moi}) (\cref{fig:car_bcma_percent}). Also consistent with CD19 and non-\gls{car} data, we also found that the number of \ptcar{} T cells was greater for \gls{dms} than for bead (\cref{fig:car_bcma_total}). \begin{figure*}[ht!] \begingroup \includegraphics{../figures/car_bcma.png} \phantomsubcaption\label{fig:car_bcma_percent} \phantomsubcaption\label{fig:car_bcma_total} \endgroup \caption[\acrshort{bcma} Transduction] {\glspl{dms} produce larger numbers of \gls{bcma} \gls{car} T cells compared to beads. \subcap{fig:car_bcma_percent}{\ptcarp{} at day 14.} \subcap{fig:car_bcma_total}{Total number of \ptcarp{} cells at day 14.} } \label{fig:car_bcma} \end{figure*} \subsection{DMSs Efficiently Expand T Cells in G-Rex Bioreactors} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/grex_results.png} \phantomsubcaption\label{fig:grex_results_fc} \phantomsubcaption\label{fig:grex_results_viability} \phantomsubcaption\label{fig:grex_mem} \phantomsubcaption\label{fig:grex_cd4} \endgroup \caption[\acrshort{grex} Expansion] {\glspl{dms} expand T cells robustly in \gls{grex} bioreactors. \subcap{fig:grex_results_fc}{Fold change of T cells over time.} \subcap{fig:grex_results_viability}{Viability of T cells over time.} \subcap{fig:grex_mem}{\ptmemp{}} and \subcap{fig:grex_cd4}{\pthp{}} of T cells after \SI{14}{\day} of expansion. Significance tests were performed using the Wilcoxon non-parametric test. } \label{fig:grex_results} \end{figure*} We also asked if the \gls{dms} platform could expand T cells in a \gls{grex} bioreactor. We incubated T cells in a \gls{grex} analogously to plates and found that T cells in \gls{grex} bioreactors expanded as efficiently as beads over \SI{14}{\day} with similar viability (\cref{fig:grex_results_fc,fig:grex_results_viability}). Consistent with past results, \glspl{dms}-expanded T cells had higher \pthp{} and \ptmemp{} compared to beads (\cref{fig:grex_mem,fig:grex_cd4}). Overall the \ptmemp{} was lower than that seen in standard plates (\cref{fig:dms_phenotype_mem}). These discrepancies might be explained in light of other data as follows. The \gls{grex} bioreactor has higher media capacity relative to its surface area, and we did not move the T cells to a larger bioreactor as they grew in contrast with our plate cultures. This means that the cells had higher growth area constraints, which may have nullified any advantage to the expansion seen in standard plates (\cref{fig:dms_exp_fold_change}). Furthermore, the higher growth area could mean increased signaling and \gls{teff} differentiation, which was why the \ptmemp{} was low compared to past data (\cref{fig:dms_phenotype_mem}). \begin{figure*}[ht!] \begingroup \includegraphics{../figures/grex_luminex.png} \endgroup \caption[\acrshort{grex} Luminex Results] {\gls{dms} lead to higher cytokine production in \gls{grex} bioreactors.} \label{fig:grex_luminex} \end{figure*} We also quantified the cytokines released during the \gls{grex} expansion using Luminex. We noted that in nearly all cases, the \gls{dms}-expanded T cells released higher concentrations of cytokines compared to beads (\cref{fig:grex_luminex}), including higher concentrations of pro-inflammatory cytokines such as \gls{gmcsf}, \gls{ifng}, and \gls{tnfa}. This demonstrates that \glspl{dms} could lead to more robust activation. Taken together, these data suggest that \gls{dms} also lead to robust expansion in \gls{grex} bioreactors, although more optimization may be necessary to maximize the media feed rate and growth area to get comparable results to those seen in tissue-culture plates. \subsection{DMSs Do Not Leave Antibodies Attached to Cell Product} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/nonstick.png} \endgroup \caption[\acrshort{dms} \acrshort{mab} Detachment] {\glspl{mab} do not detach from microcarriers onto T cells in a detectable manner. Plots are representative manufacturing runs harvest after \SI{14}{\day} of expansion and stained with \anti{\gls{igg}}. } \label{fig:nonstick} \end{figure*} We asked if \glspl{mab} from the \glspl{dms} detached from the \gls{dms} surface and could be detected on the final T cell product. This test is important for clinical translation as any residual \glspl{mab} on T cells injected into the patient could elicit an undesirable \antim{\gls{igg}} immune response. We did not detect the presence of either \ahcd{3} or \ahcd{28} \glspl{mab} (both of which were \gls{igg}) on the final T cell product after \SI{14}{\day} of expansion (\cref{fig:nonstick}). \subsection{DMSs Outperform Beads in a Variety of Conditions} In order to establish the robustness of our method, we combined all experiments performed in our lab using beads or \glspl{dms} into one dataset. Since each experiment was performed using slightly different process conditions, we hypothesized that performing causal inference on such a dataset would indicate if the \glspl{dms} indeed led to better results under a variety of conditions. The dataset was curated by compiling all experiments and filtering those that ended at day 14 and including flow cytometry results for the \ptmem{} and \pth{} populations. We further filtered our data to only include those experiments where the surface density of the CD3 and CD28 \gls{mab} were held constant (since some of our experiments varied these on the \glspl{dms}). This ultimately resulted in a dataset with 177 runs spanning 16 experiments between early 2017 and early 2021. Since the aim of the analysis was to perform causal inference, we determined 6 possible treatment variables which we controlled when designing the experiments included in this dataset. Obviously the principle treatment parameter was ``activation method'' which represented the effect of activating T cells with either beads or \glspl{dms}. We also included ``bioreactor'' which was a categorical variable for growing the T cells in a \gls{grex} bioreactor or polystyrene plates, ``feed criteria'' which represented the criteria used to feed the cells (media color or a glucose meter), ``\il{2} Feed Conc.'' as a continuous parameter for the concentration of \il{2} added each feed cycle, and ``CD19-CAR Transduced'' representing if the cells were lentivirally transduced or not. Unfortunately, many of these parameters correlated with each other despite the large size of our dataset, so the only two parameters for which causal relationships could be evaluated were ``activation method'' and ``bioreactor''. Note that these were not the only set of theoretical treatment parameters that we could have used. For example, media feed rate is an important process parameter, but in our experiments this was dependent on the feeding criteria and the growth rate of the cells, which in turn is determined by activation method. Therefore, ``media feed rate'' (or similar) is a ``post-treatment parameter,'' and including it would have violated the backdoor criteria and severely biased our estimates of the treatment parameters themselves. In addition to these treatment parameters, we also included covariates to improve the precision of our model. Among these were donor parameters including age, \gls{bmi}, demographic, and gender, as well as the initial viability and CD4:CD8 ratio of the cryopreserved cell lots used in the experiments (\cref{tab:meta_donors}). We also included the age (in days) of \il{2}, growth media, and thaw media; for \il{2} this was the time elapsed since reconstitution, and for the others it was the elapsed time since the manufacturing date according to the vendor. Each experiment was performed by one of three operators, so this was included as a three-level categorical parameter. Lastly, some of our experiments were sampled longitudinally, so we included a boolean categorical to represented this modification as removing conditioned media as the cell are expanding could disrupt signaling pathways. \begin{table}[!ht] \centering \caption{Causal inference on treatment variables} \label{tab:ci_treat} \input{../tables/causal_inference_treat.tex} \end{table} \begin{table}[!ht] \centering \caption{Causal inference on all variables} \label{tab:ci_controlled} \input{../tables/causal_inference_control.tex} \end{table} \begin{table}[!ht] \centering \caption{Causal inference on all variables (single donor)} \label{tab:ci_single} \input{../tables/causal_inference_single.tex} \end{table} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/metaanalysis_effects.png} \phantomsubcaption\label{fig:metaanalysis_fx_exp} \phantomsubcaption\label{fig:metaanalysis_fx_mem} \phantomsubcaption\label{fig:metaanalysis_fx_cd4} \endgroup \caption[Meta-analysis Effect Sizes] {\glspl{dms} exhibit superior performance compared to beads controlling for many experimental and process conditions. Effect sizes for \subcap{fig:metaanalysis_fx_exp}{fold change}, \subcap{fig:metaanalysis_fx_mem}{\ptmemp{}}, and \subcap{fig:metaanalysis_fx_cd4}{\dpthp{}}. The dotted line represents the mean of the bead population. The red and blue dots represent the effect size of using \gls{dms} instead of beads only considering treatment variables (\cref{tab:ci_treat}) or treatment and control variables (\cref{tab:ci_controlled}) respectively. } \label{fig:metaanalysis_fx} \end{figure*} We first asked what the effect of each of our treatment parameters was on the responses of interest, which were fold change of the cells, the \ptmemp{}, and \dpthp{} (\pthp{} at day 14 compared to its day 0 value). We performed a linear regression using activation method and bioreactor as predictors (the only treatments that were shown to be balanced) (\cref{tab:ci_treat}). Note that fold change was log transformed to reflect the exponential nature of T cell growth. We observe that the treatments are significant in all cases except for the \dpthp{}; however, we also observe that relatively little of the variability is explained by these simple models ($R^2$ between 0.17 and 0.44). We then included all covariates and unbalanced treatment parameters and performed linear regression again (\cref{tab:ci_controlled,fig:metaanalysis_fx}). We observe that after controlling for additional noise, the models explained much more variability ($R^2$ between 0.76 and 0.87). Furthermore, the coefficient for activation method in the case of fold change changed very little but still remained quite high (note the log-transformation) with \SI{131}{\percent} increase in fold change compared to beads. Furthermore, the coefficient for \ptmemp{} dropped to a \SI{3.5}{\percent} increase and almost became non-significant at $\upalpha$ = 0.05, and the \dpthp{} response increased to a \SI{7.4}{\percent} increase and became highly significant. Looking at the bioreactor treatment, we see that using the bioreactor in the case of fold change and \ptmemp{} is actually harmful to the response, while at the same time it seems to increase the \dpthp{} response. We should note that this parameter merely represents whether or not the choice was made experimentally to use a bioreactor or not; it does not indicate why the bioreactor helped or hurt a certain response. For example, using a \gls{grex} entails changing the cell surface and feeding strategy for the T cells, and any one of these ``mediating variables'' might actually be the cause of the responses. Finally, we stratified on the most common donor (vendor ID 338 from Astarte Biotech) as accounted for almost half the data (80 runs) and repeated the regression (\Cref{tab:ci_single}). In this case, we did not include any donor-dependent variables or any variables that were the same value for these 80 runs. In this analysis, fold change and \dpthp{} remained high (but slightly lowered from the full analysis) and \ptmemp{} was non-significant. Given this, it appears that other donors may have had high \ptmemp{}, and that high fold change and \dpthp{} may have been driven by this single donor but more extreme in other donors. \section{Discussion} We have developed a method for activating T cells which leads to superior expansion with higher number of naïve/memory and CD4+ T cells compared to state-of-the-art microbead technology (\cref{fig:dms_exp}). Other groups have used biomaterials approaches to mimic the \invivo{} microenvironment\cite{Cheung2018, Rio2018, Delalat2017, Lambert2017, Matic2013}; however, to our knowledge this is the first system that specifically drives naïve/memory and CD4+ T cell formation in a scalable, potentially bioreactor-compatible manufacturing process. Memory and naïve T cells have been shown to be important clinically. Compared to \glspl{teff}, they have a higher proliferative capacity and are able to engraft for months; thus they are able to provide long-term immunity with smaller doses\cite{Gattinoni2012, Joshi2008}. Less differentiated T cells have led to greater survival both in mouse tumor models and human patients\cite{Fraietta2018, Adachi2018, Rosenberg2011}. Furthermore, clinical response rates have been positively correlated with T cell expansion, implying that highly-proliferative naïve and memory T cells are a significant contributor\cite{Xu2014, Besser2010}. Circulating memory T cells have also been found in complete responders who received CAR T cell therapy\cite{Kalos2011}. Similarly, CD4 T cells have been shown to play an important role in CAR T cell immunotherapy. It has been shown that CAR T doses with only CD4 or a mix of CD4 and CD8 T cells confer greater tumor cytotoxicity than only CD8 T cells\cite{Wang2018, Sommermeyer2015}. There are several possible reasons for these observations. First, CD4 T cells secrete proinflammatory cytokines upon stimulation which may have a synergistic effect on CD8 T cells. Second, CD4 T cells may be less prone to exhaustion and may more readily adopt a memory phenotype compared to CD8 T cells\cite{Wang2018}. Third, CD8 T cells may be more susceptible than CD4 T cells to dual stimulation via the \gls{car} and endogenous \gls{tcr}, which could lead to overstimulation, exhaustion, and apoptosis\cite{Yang2017}. Despite evidence for the importance of CD4 T cells, more work is required to determine the precise ratios of CD4 and CD8 T cell subsets to be included in CAR T cell therapy given a disease state. When analyzing all our experiments comprehensively using causal inference, we found that all three of our responses were significantly increased when controlling for covariates (\cref{fig:metaanalysis_fx,tab:ci_controlled}). By extension, this implies that not only will \glspl{dms} lead to higher fold change overall, but also much higher fold change in absolute numbers of memory and CD4+ T cells. Furthermore, we found that using a \gls{grex} bioreactor is detrimental to fold change and memory percent while helping CD4+. Since there are multiple consequences to using a \gls{grex} compared to tissue-treated plates, we can only speculate as to why this might be the case. Firstly, when using a \gls{grex} we did not expand the surface area on which the cells were growing in a comparable way to that of polystyrene plates. One possible explanation is that the T cells spent longer times in highly activating conditions (since the beads and DMSs would have been at higher per-area concentrations in the \gls{grex} vs polystyrene plates) which has been shown to skew toward \gls{teff} populations\cite{Lozza2008}. Furthermore, the simple fact that the T cells spent more time at high surface densities could simply mean that the T cells didn’t expand as much due to spacial constraints. This would all be despite the gas-permeable membrane and tell design of the \gls{grex}, which are meant to enhance growth and not impede it. Given this, our data suggests we were using the bioreactor sub-optimally, and the hypothesized causes for why our T cells did not expand could be verified with additional experiments varying the starting cell density and/or using larger bioreactors. A key question in the space of cell manufacturing is that of donor variability. To state this precisely, this is a second order interaction effect that represents the change in effect of treatment (eg bead vs \gls{dms}) given the donor. While our meta-analysis was relatively large compared to many published experiments usually seen for technologies at this developmental stage, we have a limited ability in answering this question. We can control for donor as a covariate, and indeed our models show that many of the donor characteristics are strongly associated with each response on average, but these are first order effects and represent the association of age, gender, demographic, etc given everything else in the model is held constant. Second order interactions require that our treatments be relatively balanced and random across each donor, which is a dubious assumption for our dataset (indeed, one donor was used for nearly half of it). However, this can easily be solved by performing more experiments with these restrictions in mind, which will be a subject of future work. Furthermore, this dataset offers an interesting insight toward novel hypothesis that might be further investigated. One limitation of our dataset is that we were unable to investigate the effects of time using a method such as autoregression, and instead relied on aggregate measures such as the total amount of a reagent added over the course of the expansion. Further studies should be performed to investigate the temporal relationship between phenotype, cytokine concentrations, feed rates, and other measurements which may perturb cell cultures, as this will be the foundation of modern process control necessary to have a fully-automated manufacturing system. The \gls{dms} system could be used as a drop in replacement for beads in many of current allogeneic therapies. Indeed, given its higher potential for expansion (\cref{fig:dms_exp,tab:ci_controlled}), it may work in cases where the beads fail (although this would need to be tested by gathering data with many unhealthy donors). However, in the autologous setting patients only need a fixed dose, and thus any expansion beyond the indicated dose would be wasted. Given this, it will be interesting to apply this technology in an allogeneic paradigm where this increased expansion potential would be well utilized. While our method is superior in several ways compared to beads, the cell manufacturing field would tremendously benefit from simply having an alternative to the state-of-the-art. The licenses for bead-based expansion are controlled by few companies; having an alternative would provide more competition in the market, reducing costs and improving access for academic researchers and manufacturing companies. \chapter{AIM 2A}\label{aim2a} \section{Introduction} The purpose of this sub-aim was to develop computational methods to identify novel \glspl{cqa} and \glspl{cpp} that could be used for release criteria, process control, and process optimization for the \gls{dms} platform. We hypothesized that T cells grown using the \gls{dms} system would produce detectable biological signatures in the media supernatent which would correspond to clinically relevent responses such as fold expansion or phenotype. We tested this hypothesis by activating T cells under a variety of conditions using a \gls{doe}, sampling the media at intermediate timepoints, and creating models to predict the outcome of the cultures. We should stress that the specific \glspl{cpp} and \glspl{cqa} determined by this aim are not necessarily universal, as this was not performed with equipment that would normally be used at scale. However, the process outlined here is one that can easily be adaptable to any system, and the specific findings themselves offer interesting insights that warrant further study\footnote{adapted from \modelpaper{}}. \section{Methods} \subsection{Study Design} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/modeling_overview.png} \phantomsubcaption\label{fig:mod_overview_flow} \phantomsubcaption\label{fig:mod_overview_doe} \endgroup \caption[Modeling Overview] {Overview of modeling experiments. \subcap{fig:mod_overview_flow}{Relationship between \gls{doe} experiments and AI driven prediction. \glspl{doe} will be used to determine optimal process input conditions, and longitudinal multiomics data will be used to fit predictive models. Together, these will reveal predictive species that may be used for \glspl{cqa} and \glspl{cpp}.} \subcap{fig:mod_overview_doe}{Overview of the two \gls{doe} experiments; the initial \gls{doe} is given by the blue points and the augmented \gls{doe} is given by the blue points.} } \label{fig:mod_overview} \end{figure*} The overall workflow of this aim is shown in \cref{fig:mod_overview_flow}. Experimental conditions within the design space were explored using a \gls{doe}, and longitudinal samples were collected for each condition as the cultures progressed. Data from inputs and/or longitudinal samples were used to predict the endpoint response. The fusion of cytokine and \gls{nmr} profiles from media to model these responses included 30 cytokines from a custom Thermo Fisher ProcartaPlex Luminex kit and 20 \gls{nmr} features. These 20 spectral features from \gls{nmr} media analysis were selected out of approximately 250 peaks using a variance-based feature selection approach and some manual inspection steps. The first \gls{doe} resulted in a randomized 18-run I-optimal custom design where each \gls{dms} parameter was evaluated at three levels: \pilII{} (10, 20, and 30 U/uL), \pdms{} (500, 1500, 2500 \si{\dms\per\ml}), and \pmab{} (60, 80, 100 \si{\percent}). These 18 runs consisted of 14 unique parameter combinations where 4 of them were replicated twice to assess prediction error. To further optimize the initial region explored, an \gls{adoe} was designed with 10 unique parameter combinations, two of these replicated twice for a total of 12 additional samples (\cref{fig:mod_overview_doe}). Process parameters for the \gls{adoe} were evaluated at multiple levels: \pilII{} (30, 35, and 40 \si{\IU\per\ml}), \pdms{} (500, 1000, 1500, 2000, 2500, 3000, 3500 \si{\dms\per\ml}), and \pmab{} (\SI{100}{\percent}) (\cref{fig:mod_overview}). \subsection{DMS Fabrication} \glspl{dms} were fabricated as described in \cref{sec:dms_fab} with the following modifications in order to obtain a variable functional \gls{mab} surface density. During the \gls{mab} coating step, the \acd{3}/\acd{28} \gls{mab} mixture was further combined with a biotinylated isotype control to reduce the overall fraction of targeted \glspl{mab} (for example the \SI{60}{\percent} \gls{mab} surface density corresponded to 3 mass parts \acd{3}, 3 mass parts \acd{28}, and 4 mass parts isotype control). \subsection{T Cell Culture} T cell culture was performed as described in \cref{sec:tcellculture} with the following modifications. At days 4, 6, 8, and 11, \SI{100}{\ul} media were collected for the Luminex assay and \gls{nmr} analysis. The volume of removed media was equivalently replaced during the media feeding step, which took place immediately after sample collection. Additionally, the same media feeding schedule was followed for the \gls{doe} and \gls{adoe} to improve consistency, and the same donor lot was used for both experiments. All cell counts were performed using \gls{aopi}. \subsection{Flow Cytometry} Flow cytometry was performed analogously to \cref{sec:flow_cytometry}. \subsection{Cytokine Quantification} Cytokines were quantified via Luminex as described in \cref{sec:luminex_analysis}. \subsection{NMR Metabolomics} Prior to analysis, samples were centrifuged at \SI{2990}{\gforce} for \SI{20}{\minute} at \SI{4}{\degreeCelsius} to clear any debris\footnote{all \gls{nmr} analysis was done by our collaborators Max Colonna and Art Edison at the University of Georgia; methods included here for reference}. \SI{5}{\ul} of 100/3 \si{\mM} DSS-D6 in deuterium oxide (Cambridge Isotope Laboratories) were added to \SI{1.7}{\mm} \gls{nmr} tubes (Bruker BioSpin), followed by \SI{45}{\ul} of media from each sample that was added and mixed, for a final volume of \SI{50}{\ul} in each tube. Samples were prepared on ice in predetermined, randomized order. The remaining volume from each sample in the rack (approx. \SI{4}{\ul}) was combined to create an internal pool. This material was used for internal controls within each rack as well as metabolite annotation. \gls{nmr} spectra were collected on a Bruker Avance III HD spectrometer at \SI{600}{\MHz} using a \SI{5}{\mm} TXI cryogenic probe and TopSpin software (Bruker BioSpin). One-dimensional spectra were collected on all samples using the noesypr1d pulse sequence under automation using ICON NMR software. Two-dimensional \gls{hsqc} and \gls{tocsy} spectra were collected on internal pooled control samples for metabolite annotation. One-dimensional spectra were manually phased and baseline corrected in TopSpin. Two-dimensional spectra were processed in NMRpipe\cite{Delaglio1995}. One dimensional spectra were referenced, water/end regions removed, and normalized with the PQN algorithm\cite{Dieterle2006} using an in-house MATLAB (The MathWorks, Inc.) toolbox. To reduce the total number of spectral features from approximately 250 peaks and enrich for those that would be most useful for statistical modeling, a variance-based feature selection was performed within MATLAB. For each digitized point on the spectrum, the variance was calculated across all experimental samples and plotted. Clearly-resolved features corresponding to peaks in the variance spectrum were manually binned and integrated to obtain quantitative feature intensities across all samples. In addition to highly variable features, several other clearly resolved and easily identifiable features were selected (glucose, \gls{bcaa} region, etc). Some features were later discovered to belong to the same metabolite but were included in further analysis. Two-dimensional spectra collected on pooled samples were uploaded to COLMARm web server, where \gls{hsqc} peaks were automatically matched to database peaks. \gls{hsqc} matches were manually reviewed with additional 2D and proton spectra to confirm the match. Annotations were assigned a confidence score based upon spectral data levels supporting the match as previously described\cite{Dashti2017}. Annotated metabolites were matched to previously selected features used for statistical analysis. Several low abundance features selected for analysis did not have database matches and were not annotated. Statistical total correlation spectroscopy\cite{Holmes2006} suggested that some of these unknown features belonged to the same molecules (not shown). Additional multidimensional \gls{nmr} experiments will be required to determine their identity. \subsection{Machine Learning and Statistical Analysis} Linear regression analysis of the \glspl{doe} was performed as described in \cref{sec:statistics}. Seven \gls{ml} techniques were implemented to predict three responses related to the memory phenotype of the cultured T cells under different process conditions (\rmemh{}, \rmemk{}, and \rratio{}). The \gls{ml} methods executed were \gls{rf}, \gls{gbm}, \gls{cif}, \gls{lasso}, \gls{plsr}, \gls{svm}, and DataModeler’s \gls{sr}\footnote{\gls{sr} was performed by Theresa Kotanchek at Evolved Analytics, \gls{rf}, \gls{gbm}, \gls{cif}, \gls{plsr}, \gls{svm} were performed by Valerie Odeh-Couvertier at UPRM. Methods included here for reference}. Primarily, \gls{sr} models were used to optimize process parameter values based on \ptmem{} phenotype and to extract early predictive variable combinations from the multi-omics experiments. Furthermore, high-performing models from each method were used in consensus analysis to extract potential \glspl{cqa} and \glspl{cpp} predictive of T cell potency, safety, and consistency at the early stages of the manufacturing process. \gls{sr} was done using Evolved Analytics’ DataModeler software (Evolved Analytics LLC, Midland, MI). DataModeler utilizes genetic programming to evolve symbolic regression models (both linear and non-linear) rewarding simplicity and accuracy. Using the selection criteria of highest accuracy ($R^2>\SI{90}{\percent}$) and lowest complexity, the top-performing models were identified. Driving variables, variable combinations, and model dimensionality tables were generated. The top-performing variable combinations were used to generate model ensembles. In this analysis, DataModeler’s \inlinecode{SymbolicRegression} function was used to develop explicit algebraic (linear and nonlinear) models. The fittest models were analyzed to identify the dominant variables using the \inlinecode{VariablePresence} function, the dominant variable combinations using the \inlinecode{VariableCombinations} function, and the model dimensionality (number of unique variables) using the \inlinecode{ModelDimensionality} function. \inlinecode{CreateModelEnsemble} was used to define trustable model ensembles using selected variable combinations and these were summarized (model expressions, model phenotype, model tree plot, ensemble quality, model quality, variable presence map, \gls{anova} tables, model prediction plot, exportable model forms) using the \inlinecode{ModelSummaryTable} function. Ensemble prediction and residual performance were assessed via the \inlinecode{EnsemblePredictionPlot} and \inlinecode{EnsembleResidualPlot} subroutines respectively. Model maxima (\inlinecode{ModelMaximum} function) and model minima (\inlinecode{ModelMinimum} function) were calculated and displayed using the \inlinecode{ResponsePlotExplorer} function. Trade-off between multiple responses was explored using \inlinecode{MultiTargetResponseExplorer} and \inlinecode{ResponseComparisonExplorer} with additional insights derived from \inlinecode{ResponseContourPlotExplorer}. Graphics and tables were generated by DataModeler. These model ensembles were used to identify predicted response values, potential optima in the responses, and regions of parameter values where the predictions diverge the most. Non-parametric tree-based ensembles were done through the \inlinecode{randomForest}, \inlinecode{gbm}, and \inlinecode{cforest} regression functions in R, for \gls{rf}, \gls{gbm}, and \gls{cif} models, respectively. Both \gls{rf} and \gls{cif} construct multiple decision trees in parallel, by randomly choosing a subset of features at each decision tree split, in the training stage. \gls{rf} individual decision trees are split using the Gini Index, while conditional inference forest uses a statistical significance test procedure to select the variables at each split, reducing correlation bias. In contrast, \gls{gbm} construct regression trees in series through an iterative procedure that adapts over the training set. This model learns from the mistakes of previous regression trees in an iterative fashion to correct errors (\gls{mse}) from its precursors’ trees. Prediction performance was evaluated using \gls{loocv} and permutation-based variable importance scores assessing percent increase of \gls{mse}, relative influence based on the increase of prediction error, coefficient values for \gls{rf}, \gls{gbm}, and \gls{cif}, respectively. \gls{plsr} was executed using the \inlinecode{plsr} function from the \inlinecode{pls} package in R while \gls{lasso} regression was performed using the \inlinecode{cv.glmnet} R package, both using \gls{loocv}. Finally, the \inlinecode{kernlab} R package was used to construct the \gls{svm} models. Parameter tuning was done for all models in a grid search manner using the train function from the \inlinecode{caret} R package using \gls{loocv} as the optimization criteria. Specifically, the number of features randomly sampled as candidates at each split (\inlinecode{mtry}) and the number of trees to grow (\inlinecode{ntree}) were tuned parameters for random forest and conditional inference forest. In particular, minimum sum of weights in a node to be considered for splitting and the minimum sum of weights in a terminal node were manually tuned for building the \gls{cif} models. Moreover, \gls{gbm} parameters such as the number of trees to grow, maximum depth of each tree, learning rate, and the minimal number of observations at the terminal node, were tuned for optimum \gls{loocv} performance as well. For \gls{plsr}, the optimal number of components to be used in the model was assessed based on the standard error of the cross-validation residuals using the function \inlinecode{selectNcomp} from the \inlinecode{pls} package. Moreover, \gls{lasso} regression was performed using the \inlinecode{cv.glmnet} package with $\upalpha$ = 1. The best $\uplambda$ for each response was chosen using the minimum error criteria. Lastly, a fixed linear kernel (\inlinecode{svmLinear}) was used to build the \gls{svm} regression models evaluating the cost parameter value with best \gls{loocv}. Prediction performance was measured for all models using the final model with \gls{loocv} tuned parameters. \subsection{Consensus Analysis} Consensus analysis of the relevant variables extracted from each machine learning model was done to identify consistent predictive features of quality at the early stages of manufacturing. First, importance scores for all features were measured across all \gls{ml} models using \inlinecode{varImp} with \inlinecode{caret} R package except for scores for \gls{svm} which \inlinecode{rminer} R package was used. These importance scores were percent increase in \gls{mse}, relative importance through average increase in prediction error when a given predictor is permuted, permuted coefficients values, absolute coefficient values, weighted sum of absolute coefficients values, and relative importance from sensitivity analysis determined for \gls{rf}, \gls{gbm}, \gls{cif}, \gls{lasso}, \gls{plsr}, and \gls{svm}, respectively. Using these scores, key predictive variables were selected if their importance scores were within the \nth{80} percentile ranking for the following \gls{ml} methods: \gls{rf}, \gls{gbm}, \gls{cif}, \gls{lasso}, \gls{plsr}, \gls{svm} while for \gls{sr} variables present in >\SI{30}{\percent} of the top-performing \gls{sr} models from DataModeler ($R^2\ge \SI{90}{\percent}$, Complexity $\ge 100$) were chosen to investigate consensus except for \gls{nmr} media models at day 4 which considered a combination of the top-performing results of models excluding lactate ppms, and included those variables which were in >\SI{40}{\percent} of the best performing models. Only variables with high percentile scoring values were evaluated in terms of their logical relation (intersection across \gls{ml} models) and depicted using a Venn diagram from the \inlinecode{venn} R package. \section{Results} \subsection{DMSs Grow T Cells With Lower IL2 Concentrations} Prior to the main experiments in this aim, we assessed the effect of lowering the \il{2} concentration on the T cells grown with either bead or \gls{dms}. One of our hypotheses for the \gls{dms} system was that higher cell density would enhance cross-talk between T cells. Since \il{2} is secreted by activated T cells themselves, T cells in the \gls{dms} system may need less or no \il{2} if this is true. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/il2_modulation.png} \phantomsubcaption\label{fig:il2_mod_timecourse} \phantomsubcaption\label{fig:il2_mod_total} \phantomsubcaption\label{fig:il2_mod_mem} \phantomsubcaption\label{fig:il2_mod_flow} \endgroup \caption[T Cells Grown at Varying IL2 Concentrations] {\glspl{dms} grow T cells effectively at lower \il{2} concentrations. \subcap{fig:il2_mod_timecourse}{Longitudinal cell counts of T cells grown with either bead or \glspl{dms} using varying \il{2} concentrations.} Day 14 counts of either \subcap{fig:il2_mod_total}{total cells} or \subcap{fig:il2_mod_mem}{\ptmem{} cells} plotted against \il{2} concentration. \subcap{fig:il2_mod_flow}{Flow cytometry plots of the \ptmem{} gated populations at day 14 of culture for each \il{2} concentration.} } \label{fig:il2_mod} \end{figure*} We varied the concentration of \il{2} from \SIrange{0}{100}{\IU\per\ml} and expanded T cells as described in \cref{sec:tcellculture}. T cells grown with either method expanded robustly as \il{2} concentration was increased (\cref{fig:il2_mod_timecourse}). Surprisingly, neither the bead or the \gls{dms} group expanded at all with \SI{0}{\IU\per\ml} \il{2}. When examining the endpoint fold change after \SI{14}{\day}, we observed that the difference between the bead and \gls{dms} appears to be greater at lower \il{2} concentrations (\cref{fig:il2_mod_total}). Furthermore, the same trend can be seen when only examining the \ptmem{} cell expansion at day 14 (\cref{fig:il2_mod_mem}). In this case, the \ptmemp{} of the T cells seemed to be relatively close at higher \il{2} concentrations, but separated further at lower concentrations (\cref{fig:il2_mod_flow}) Taken together, these data do not support the hypothesis that the \gls{dms} system does not need \il{2} at all; however, it appears to have a modest advantage at lower \il{2} concentrations compared to beads. For this reason, we decided to investigate the lower range of \il{2} concentrations starting at \SI{10}{\IU\per\ml} in the remainder of this aim. \subsection{DOE Shows Optimal Conditions for Potent T Cells} \begin{table}[!ht] \centering \begin{threeparttable} \caption{DOE Runs} \label{tab:doe_runs} \input{../tables/doe_runs.tex} \begin{tablenotes} \item[a] It was determined later that the total \glspl{mab} surface density may not be consistent across each batch of \gls{dms} used. Thus, these runs were taken out as they were created at different scale and with a different operator compared to the rest. Leaving them in may produce unobserved confounding factors \end{tablenotes} \end{threeparttable} \end{table} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/doe_responses_first.png} \phantomsubcaption\label{fig:doe_response_first_mem} \phantomsubcaption\label{fig:doe_response_first_cd4} \endgroup \caption[Response Plots for First \acrshort{doe}] {Response plots from the first \gls{doe} experiment for \subcap{fig:doe_response_first_mem}{\ptmemp{}} and \subcap{fig:doe_response_first_cd4}{\pthp{}}. Each point is one run. } \label{fig:doe_response_first} \end{figure*} We conducted two consecutive \glspl{doe} to optimize the \pth{} and \ptmem{} responses for the \gls{dms} system. In the first, we tested \pilII{} in the range of \SIrange{10}{30}{\IU\per\ml}, \pdms{} in the range of \SIrange{500}{2500}{\dms\per\ml}, and \pmab{} in the range of \SIrange{60}{100}{\percent}. When looking at total \ptmemp{} cells, \pilII{} showed a positive linear trend and \pdms{} and \pmab{} showed possible second-order effects with intermediate maximums and minimums respectively (\cref{fig:doe_response_first_mem}). In the case of \pth{}, all parameters showed a positive, suggesting a maximum might exist at a higher value for each. After performing the first \gls{doe}, we augmented the original design matrix with an \gls{adoe} which was built with three goals in mind. Firstly we wished to validate the first \gls{doe} by assessing the strength and responses of each effect. Secondly, we wished to improve our confidence in regions that showed high complexity, such as the peak in the \gls{dms} concentration for the total \ptmem{} cell response. Thirdly, we wished to explore additional ranges of each response. Notably, \pilII{} appeared to increase beyond our tested range, thus we were curious if there was an optimum at some higher setting. For this reason, we increased the \pilII{} to include \SI{40}{\IU\per\ml} and the \pdms{} to \SI{3500}{\dms\per\ml}. Note that it was impossible to go beyond \SI{100}{\percent} for the \pmab{}, so runs were positioned for this parameter with validation and confidence improvements in mind. The runs for each \gls{doe} were shown in \cref{tab:doe_runs}. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/doe_responses.png} \phantomsubcaption\label{fig:doe_responses_mem} \phantomsubcaption\label{fig:doe_responses_cd4} \phantomsubcaption\label{fig:doe_responses_mem4} \phantomsubcaption\label{fig:doe_responses_ratio} \endgroup \caption[T Cell Optimization Through \acrshortpl{doe}] {\gls{doe} methodology reveals optimal conditions for expanding T cell subsets. Responses vs \il{2} concentration, \gls{dms} concentration, and functional \gls{mab} percentage are shown for \subcap{fig:doe_responses_mem}{total \ptmem{} T cells}, \subcap{fig:doe_responses_cd4}{total \pth{} T cells}, \subcap{fig:doe_responses_mem4}{total \ptmemh{} T cells}, and \subcap{fig:doe_responses_ratio}{ratio of CD4 and CD8 T cells in the \ptmem{} compartment}. Each point represents one run. } \label{fig:doe_responses} \end{figure*} \begin{table}[!ht] \centering \caption{Regression for total \ptmem{} cells (first order)} \label{tab:doe_mem1.tex} \input{../tables/doe_mem1.tex} \end{table} \begin{table}[!ht] \centering \caption{Regression for total \ptmem{} cells (third order)} \label{tab:doe_mem2.tex} \input{../tables/doe_mem2.tex} \end{table} \begin{table}[!ht] \centering \caption{Regression for total \pth{} cells} \label{tab:doe_cd4.tex} \input{../tables/doe_cd4.tex} \end{table} \begin{table}[!ht] \centering \caption{Regression for total \ptmemh{} cells} \label{tab:doe_mem4.tex} \input{../tables/doe_mem4.tex} \end{table} \begin{table}[!ht] \centering \caption{Regression for \ptmem{} CD4:CD8 ratio} \label{tab:doe_ratio.tex} \input{../tables/doe_ratio.tex} \end{table} The response plots from both \glspl{doe} are shown in \cref{fig:doe_responses} for total \ptmem{} cells, total \pth{} cells, total \ptmemh{} cells, and CD4:CD8 ratio in the \ptmem{} compartment. In general, the responses for the first and second \gls{doe} seemed to overlap, although not perfectly. Interestingly, only the \ptmem{} response seemed to have anything more complex than a linear relationship, particularly in the case of \pilII{} and \pdms{}, which showed intermediate optimums (\cref{fig:doe_responses_mem}). In the case of \pilII{}, it was not clear if this optimum was simply due to a batch effect of being from the first or second \gls{doe}. The optimum for \pdms{} appeared in the same location albeit more pronounced in the second \gls{doe} so, giving more confidence to the location of this second order feature. The remainder of the responses showed mostly linear relationships in all parameter cases (\cref{fig:doe_responses_cd4,fig:doe_responses_mem4,fig:doe_responses_ratio}). We performed linear regression on the three input parameters as well as a binary parameter representing if a given run came from the first or second \gls{doe} (called ``dataset''). Starting with the total \ptmem{} cells response, we fit a first order regression model using these four parameters (\cref{tab:doe_mem1.tex}). While \pilII{} was found to be a significant predictor, the model fit was extremely poor ($R^2 = 0.331$). This was not surprising given the apparent complexity of this response (\cref{fig:doe_responses_mem}). To obtain a better fit, we added second and third degree terms (\cref{tab:doe_mem2.tex}). Note that the dataset parameter was not included in the second order interaction as this was treated as a blocking variable, which are typically not assumed to have interaction effects. Also note that the response was log-transformed, which yielded a better fit. In this model many more parameters emerged as being significant, including the quadratic terms for \pdms{} and \pilII{}, in agreement with what can be qualitatively observed in the response plot (\cref{fig:doe_responses_mem}). Furthermore, the dataset parameter was weakly significant, indicating a possible batch effect between the \glspl{doe}. We should also note that despite many parameters being significant, this model was still only mediocre in describing this response; the $R^2$ was 0.741 but the $R_{adj}^2$ was 0.583, indicating that our data might be underpowered for a model this complex. Further experiments beyond what was performed here may be needed to fully describe this response. We performed linear regression on the other three responses, all of which performed much better than the \ptmem{} response as expected given the lower apparent complexity in the response plots (\cref{fig:doe_responses_cd4,fig:doe_responses_mem4,fig:doe_responses_ratio}). All these models appeared to fit will, with $R^2$ and $R_{adj}^2$ upward of 0.8. In all but the CD4:CD8 \ptmem{} ratio, the dataset parameter emerged as significant, indicating a batch effect between the \glspl{doe}. All other parameters except \pilII{} in the case of CD4:CD8 \ptmem{} ratio were significant predictors. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/doe_sr_contour.png} \phantomsubcaption\label{fig:doe_sr_contour_mem4} \phantomsubcaption\label{fig:doe_sr_contour_ratio} \endgroup \caption[Contour Plots for \acrshort{doe} Responses] {Symbolic regression and contour plots reveal optimal conditions for \subcap{fig:doe_sr_contour_mem4}{\ptmemh{} cells} and \subcap{fig:doe_sr_contour_ratio}{CD4:CD8 ratio in the \ptmem{} compartment}. } \label{fig:doe_sr_contour} \end{figure*} We then visualized the total \ptmemh{} cells and \rratio{} using the response explorer in DataModeler to create contour plots around the maximum responses. For both, maximizing all input parameters maximized both responses (\cref{fig:doe_sr_contour}). While not all combinations at and around this optimum were tested, these plots suggest that there were no other optimal values elsewhere. \subsection{Modeling with Machine Learning Reveals Putative CQAs} Due to the heterogeneity of the multivariate data collected and knowing that no single model is perfect for all applications, we implemented an agnostic modeling approach to better understand these \ptmem{} responses. To achieve this, a consensus analysis using seven \gls{ml} techniques, \gls{rf}, \gls{gbm}, \gls{cif}, \gls{lasso}, \gls{plsr}, \gls{svm}, and DataModeler’s \gls{sr}, was implemented to molecularly characterize \ptmem{} cells and to extract predictive features of quality early in their expansion process. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/doe_luminex.png} \endgroup \caption[Cytokine Release Profile of T Cells from \acrshort{doe}] {T cells show robust and varying cytokine responses over time} \label{fig:doe_luminex} \end{figure*} We collected secretome data via luminex for days 4, 6, 8, 11, and 14. Plotting the concentrations of these cytokines showed a large variation over all runs and between different timepoints, demonstrating that these could be used to differentiate between different process conditions qualitatively simply based on variance (\cref{fig:doe_luminex}). These were also much higher in most cases that a set of bead based runs which were run in parallel, in agreement with the luminex data obtained previously in the \gls{grex} system (these data were collected in plates) (\cref{fig:grex_luminex}). \begin{table}[!ht] \centering \caption[Machine Learning Model Results] {Results for \gls{ml} modeling using process parameters (PP) with only \gls{nmr} on day 4 (N4), only \gls{nmr} on day 6 (N6), only secretome on day 6 (S6), or various combindation of each for all seven \gls{ml} techniques} \label{tab:mod_results} \input{../tables/model_results.tex} \end{table} \gls{sr} models achieved the highest predictive performance ($R^2>\SI{93}{\percent}$) when using multi-omics predictors for all endpoint responses (\cref{tab:mod_results}). \gls{sr} achieved $R^2>\SI{98}{\percent}$ while \gls{gbm} ensembles showed \gls{loocv} $R^2>\SI{95}{\percent}$ for \rmemh{} and \rmemk{} responses. Similarly, \gls{lasso}, \gls{plsr}, and \gls{svm} methods showed consistently high \gls{loocv}, (\SI{92.9}{\percent}, \SI{99.7}{\percent}, and \SI{90.5}{\percent} respectively), to predict the \rratio{}. Yet, about \SI{10}{\percent} reduction in \gls{loocv}, \SIrange{72.5}{81.7}{\percent}, was observed for \rmemh{} with these three methods. Lastly, \gls{sr} and \gls{plsr} achieved $R^2>\SI{90}{\percent}$ while other \gls{ml} methods exhibited exceedingly variable \gls{loocv} (\SI{0.3}{\percent} for \gls{rf} to \SI{51.5}{\percent} for \gls{lasso}) for \rmemk{}. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/sr_omics.png} \endgroup \caption[Symbolic Regression Cytokine Dependencies] {Multi-omics culturing media prediction profiles at day 6 using symbolic regression.} \label{fig:sr_omics} \end{figure*} The top-performing technique, \gls{sr}, showed that the median aggregated predictions for \rmemh{} \rmemk{} increases when \il{2} concentration, \il{15}, and \ilr{2} increase while \il{17a} decreases in conjunction with other features. These patterns combined with low values of \pdms{} and \gls{gmcsf} uniquely characterized maximum \rmemk{}. Meanwhile, higher glycine but lower \il{13} in combination with others showed maximum \rmemh{} predictions (\cref{fig:sr_omics}). \begin{figure*}[ht!] \begingroup \includegraphics{../figures/modeling_flower.png} \phantomsubcaption\label{fig:mod_flower_48r} \phantomsubcaption\label{fig:mod_flower_cd4} \endgroup \caption[\acrshort{cqa} Consensus Plots] {Data-driven modeling using techniques with regularization reveals species predictive species which are candidates for \glspl{cqa}. Flower plots are shown for \subcap{fig:mod_flower_48r}{CD4:CD8 ratio} and \subcap{fig:mod_flower_cd4}{total \ptmemh{} cells}. The left and right columns includes models that were trained only on the secretome and metabolome respectively. Each flower on each plot represents one model, moving toward the center indicates higher agreement between models.} \label{fig:mod_flower} \end{figure*} Selecting \gls{cpp} and \glspl{cqa} candidates consistently for T cell memory is desired. Here, \gls{tnfa} was found in consensus across all seven \gls{ml} methods for predicting \rratio{} when considering features with the highest importance scores across models (\cref{fig:mod_flower_48r}). Other features, \ilr{2}, \il{4}, \il{17a}, and \pdms{}, were commonly selected in $\ge$ 5 \gls{ml} methods (\cref{fig:mod_flower_48r}). When restricting the models only to include metabolome, formate was the sole predictor shared by all. When performing similar analysis on \rmemh{}, no species for either secretome or metabolome was shared by all models (\cref{fig:mod_flower_cd4}). These models also had worse fits compared to those for \rratio{} (\cref{tab:mod_results}). For the secretome, \il{4}, \il{17a}, and \ilr{2} were agreed upon by $\ge$ 5 models. For the metabolome, formate once again was shared by $\ge$ 5 models as well as lactate. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/nmr_cors.png} \phantomsubcaption\label{fig:nmr_cors_lactate} \phantomsubcaption\label{fig:nmr_cors_formate} \phantomsubcaption\label{fig:nmr_cors_glucose} \phantomsubcaption\label{fig:nmr_cors_matrix} \endgroup \caption[NMR Day 4 Correlations] {\gls{nmr} features at day 4 are strongly correlated with each other and the response. Highly correlated relationships are shown for \subcap{fig:nmr_cors_lactate}{lactate}, \subcap{fig:nmr_cors_formate}{formate}, and \subcap{fig:nmr_cors_glucose}{glucose}. Blue and red connections indicate positive and negative correlations respectively. The threshold for visualizing connections in all cases was 0.8. \subcap{fig:nmr_cors_matrix}{Correlation matrix for all features and total \ptmemh{} yield.} } \label{fig:nmr_cors} \end{figure*} We also asked if day 4 \gls{nmr} features could predict \ptmemh{}; these models generally fit well despite being 2 days earlier in the process (\cref{fig:nmr_cors}). Lactate and formate correlated with each other and \rmemh{}. Furthermore, lactate positively correlated with \pdms{} and negatively correlated with glucose (\cref{fig:nmr_cors_lactate}). Formate also had the same correlation patterns (\cref{fig:nmr_cors_formate}). Glucose was only negatively correlated with formate and lactate (\cref{fig:nmr_cors_glucose}). Together, these data suggest that lactate, formate, \pdms{}, and \rmemh{} are fundamentally linked. \section{Discussion} \gls{cpp} modeling and understanding are critical to new product development and have life-saving implications in the context of cell therapy. The challenges for effective modeling grow with the increasing process complexity due to high dimensionality, interactions between parameters, nonlinearity. Another critical challenge is the limited amount of available data. \gls{sr} has the necessary capabilities to resolve the issues of process effects modeling and has been applied across multiple industries\cite{Kordona}. \gls{sr} discovers mathematical expressions that fit a given sample and differs from conventional regression techniques in that a model structure is not defined \textit{a priori}\cite{Koza1992}. Hence, a key advantage of this methodology is that transparent, human-interpretable models can be generated from small and large datasets with few prior assumptions\cite{Kotancheka}. Since the model search process lets the data determine the model, diverse and competitive model structures are typically discovered. An diverse ensemble will contain models that agree in regions constrained by observable data and diverge in regions without data. Collecting data in divergent regions ensures the system is accurately modeled and its optimum accurately located\cite{Kotancheka}. Consequently, this \gls{adoe} approach is useful in a many scenarios, including maximizing model validity for model-based decision making, optimizing processing parameters to maximize yield, and developing emulators for online optimization and human understanding\cite{Kotancheka}. An in-depth characterization of potential \gls{dms}-based T cell \glspl{cqa} includes a list of cytokine and \gls{nmr} features from media samples that are crucial to fate and effector functions of immune cells. Cytokine features slightly improved prediction and dominated the ranking of important features and variable combinations when modeling together with \gls{nmr} media analysis and process parameters (\cref{fig:mod_flower}). Predictive cytokine features such as \gls{tnfa}, \ilr{2}, \il{4}, \il{17a}, \il{13}, and \il{15} were biologically assessed in terms of their known functions and activities associated with T cells. T helper cells secrete more cytokines than T cytotoxic cells, as per their main functions, and activated T cells secrete more cytokines than resting T cells. It is possible that some cytokines simply reflect the \rratio{} and the activation degree by proxy proliferation. However, the exact ratio of expected cytokine abundance is less clear and depends on the subtypes present, thus examination of each relevant cytokine is needed. \ilr{2} is secreted by activated T cells and binds to \il{2}, acting as a sink to dampen its effect on T cells\cite{Witkowska2005}. Since \ilr{2} was more abundant than \il{2} in solution, this might reduce the overall effect of \il{2}, which could be further investigated by blocking \ilr{2} with an antibody. In T cells, \gls{tnfa} can increase \ilr{2}, proliferation, and cytokine production\cite{Mehta2018}. It may also induce apoptosis depending on concentration and alter the CD4:CD8 ratio\cite{Vudattu2005}. Given that TNF has both a soluble and membrane-bound form, this may either increase or decrease CD4:CD8 ratio and/or memory T cells depending on the ratio of the membrane to soluble TNF\cite{Mehta2018}. Since only soluble \gls{tnfa} was measured, membrane \gls{tnfa} is needed to understand its impact on both CD4:CD8 ratio and memory T cells. Furthermore, \il{13} is known to be critical for \gls{th2} response and therefore could be secreted if there are significant \glspl{th2} already present in the starting population\cite{Wong2011}. This cytokine has limited signaling in T cells and is thought to be more of an effector than a differentiation cytokine\cite{Junttila2018}. It might be emerging here due to an initially large number of \glspl{th2} or because \glspl{th2} were preferentially expanded; indeed, \il{4}, also found important, is the canonical cytokine that induces \gls{th2} differentiation (\cref{fig:mod_flower}). The role of these cytokines could be investigated by quantifying \glspl{th1}, \glspl{th2}, or \glspl{th17} both in the starting population and longitudinally. Similar to \il{13}, \il{17} is an effector cytokine produced by \glspl{th17}\cite{Amatya2017} thus may reflect the number of \glspl{th17} in the population. \gls{gmcsf} has been linked with activated T cells, specifically \glspl{th17}, but it is not clear if this cytokine is inducing differential expansion of CD8+ T cells or if it is simply a covariate with another cytokine inducing this expansion\cite{Becher2016}. Finally, \il{15} has been shown to be essential for memory signaling and effective in skewing \gls{car} T cells toward \glspl{tscm} when using membrane-bound \ilXVra{} and \ilr{15}\cite{Hurton2016}. Its high predictive behavior goes with its ability to induce large numbers of memory T cells by functioning in an autocrine/paracrine manner and could be explored by blocking either the cytokine or its receptor. Moreover, many predictive metabolites found here are consistent with metabolic activity associated with T cell activation and differentiation, yet it is not clear how the various combinations of metabolites relate with each other in a heterogeneous cell population. Formate and lactate were found to be highly predictive and observed to positively correlate with higher values of total live \rmemh{} cells (\cref{fig:nmr_cors}). Formate is a byproduct of the one-carbon cycle implicated in promoting T cell activation\cite{RonHarel2016}. Importantly, this cycle occurs between the cytosol and mitochondria, from which formate is excreted\cite{Pietzke2020}. Mitochondrial biogenesis and function are shown to be necessary for memory cell persistence\cite{van_der_Windt_2012, Vardhana2020}. Therefore, increased formate in media could be an indicator of one-carbon metabolism and mitochondrial activity in the culture. In addition to formate, lactate was found as a putative \gls{cqa} of \ptmem{} cells. Lactate is the end-product of aerobic glycolysis, characteristic of highly proliferating cells and activated T cells\cite{Lunt2011, Chang2013}. Glucose import and glycolytic genes are upregulated in response to T cell stimulation, thus leading to lactate. At earlier time-points, this abundance suggests a more robust induction of glycolysis and higher overall T cell proliferation. Interestingly, our models indicate that higher lactate predicts higher CD4+, both in total and in proportion to CD8+, seemingly contrary to previous studies showing that CD8+ T cells rely more on glycolysis for proliferation following activation\cite{Cao2014}. It may be that glycolytic cells dominate in the culture at the early time points used for prediction, and higher lactate reflects more cells. Metabolites that consistently decreased over time are consistent with the primary carbon source (glucose) and essential amino acids (\gls{bcaa}, histidine) that must be continually consumed by proliferating cells. Moreover, the inclusion of glutamine in our predictive models also suggests the importance of other carbon sources for certain T cell subpopulations. Glutamine can be used for oxidative energy metabolism in T cells without the need for glycolysis\cite{Cao2014}. Overall, these results are consistent with existing literature that show different T cell subtypes require different relative levels of glycolytic and oxidative energy metabolism to sustain the biosynthetic and signaling needs of their respective phenotypes\cite{Almeida2016,Wang_2012}. It is worth noting that the trends of metabolite abundance here are potentially confounded by the partial replacement of media that occurred periodically during expansion, thus likely diluting some metabolic byproducts (such as formate, lactate) and elevating depleted precursors (such as glucose and amino acids). More definitive conclusions of metabolic activity across the expanding cell population can be addressed by a closed system, ideally with on-line sensors and controls for formate, lactate, ethanol, and glucose. Practically, knowledge of how cytokines and/or metabolites are related to outcome can be utilized for process control, which involves measuring the current state of the culture, comparing it to a desired state, and intervening if it is outside an acceptable range. In the case of lactate and formate, a benchtop \gls{nmr} can be tuned to quantify lactate and formate to sample the media in real time during culture. Formate is part of the one-carbon pathway, and thus culture fate may be controlled by altering the inputs to this pathway (glycine, serine, choline) and/or adding folic acid inhibitors\cite{Ducker2017}. Since lactate is a direct byproduct of glycolysis, this may be controlled by altering the concentration of glucose in solution. Each of these control schemes would need further study to assess if they have enough precision and temporal resolution to reasonably ensure product quality. For cytokines, there is currently no analogue to a benchtop \gls{nmr}; however, research is underway to develop protein-specific sensors using aptamers\cite{Parolo2020}. Even without these developments, \gls{elisa} or Luminex can still quantify cytokines in a semi-automated manner. However, these are temporally discrete and impose a non-trivial delay before the intervention can be performed. \chapter{AIM 2B}\label{aim2b} \section{Introduction} The purpose of this sub-aim was to further explore the \gls{dms} platform, specifically for mechanisms and pathways that could be the basis for additional \glspl{cpp} that could be optimized to yield higher quantity and quality of T cells. Our strategy in general was to perturb the \gls{dms} system from the normal operating conditions at which it was used up until this point either through temporal modulation of activation signal or by blocking pathways of interest using \glspl{mab}. \section{Methods} \subsection{DMSs Temporal Modulation} \glspl{dms} were digested in active T cell cultures via addition of sterile \product{\gls{colb}}{\sigald}{11088807001} or \product{\gls{cold}}{\sigald}{11088858001}. Collagenase was dissolved in \product{\gls{hbss}}{Gibco}{14025-076} or \product{TexMACS}{\miltenyi}{170-076-307} at approximately \SI{100}{\ug\per\ml}. This solution was added to T cell cultures at a 1:1 ratio in place of plain media normally used to feed the cells during the regular media addition cycle at day 4. Cultures were then incubated as described in \cref{sec:tcellculture}, and the \glspl{dms} were verified to have been digested after \SI{24}{\hour}. Adding \glspl{dms} was simpler; the number of \gls{dms} used per area on day 0 was scaled up by 3 on day 4 to match the change from a 96 well plate to a 24 well plate, effectively producing a constant activation signal. \subsection{Mass Cytometry and Clustering Analysis} T cells were stained using a \product{34 \gls{cytof} marker panel}{Fluidigm}{201322} and \product{cisplatin}{Fluidigm}{201064} which were used according to the manufacturer’s instructions. \numrange{2e6}{3e6} stained cells per group were analyzed on a Fluidigm Helios. Unbiased cell clusters were obtained using \gls{spade} analysis by pooling three representative \gls{fcs} files and running \gls{spade} with k-means clustering (k = 100), arcsinh transformation with cofactor 5, density calculation neighborhood size of 5, local density approximation factor of 1.5, target density of 20000 cells, and outlier density cutoff of \SI{1}{\percent}\cite{Qiu2017}. All markers in the \gls{cytof} panel were used in the analysis \subsection{Integrin Blocking Experiments} To block \gls{a2b1} and \gls{a2b2}, active T cell cultures with \gls{dms} were supplemented with \product{\anti{\gls{a2b1}}}{\sigald}{MAB1973Z} and \product{\anti{\gls{a2b2}}}{\sigald}{MAB1950Z} (both \gls{leaf}) at indicated concentrations and timepoints. T cells were grown as described in \cref{sec:tcellculture}. \gls{a2b1} and \gls{a2b2} were verified to be present on active T cell cultures by staining with \product{\anti{\gls{a2b1}}-\gls{apc}}{\bl}{328313} and \product{\anti{\gls{a2b2}}-\gls{fitc}}{\bl}{359305} on day 6 of culture and analyzing via a \bd{} Accuri flow cytometer. \subsection{IL15 Blocking Experiments} To block the \ilXVra{}, we supplemented T cell cultures activated with \gls{dms} with either \product{\anti{\ilXVra{}}}{RnD}{AF247} or \product{\gls{igg} isotype control}{RnD}{AB-108-C} at the indicated timepoints and concentrations. T cells were grown as otherwise described in \cref{sec:tcellculture} with the exception that volumes were split by $\frac{1}{3}$ to keep the culture volume constant and minimize the amount of \gls{mab} required. To block soluble \il{15}, we supplemented analogously with \product{\anti{\il{15}}}{RnD}{EEP0419081} or \product{\gls{igg} isotype control}{\bl}{B236633}. \section{Results} \subsection{Adding or Removing DMSs Alters Expansion and Phenotype} We hypothesized that adding or removing \gls{dms} in the middle of an active culture would alter the activation signal and hence the growth trajectory and phenotype of T cells. While adding \glspl{dms} was simple, the easiest way to remove \glspl{dms} was to use enzymatic digestion. Collagenase is an enzyme that specifically targets collagen proteins. Since our \glspl{dms} are composed of porcine-derived collagen, this enzyme should target the \gls{dms} while sparing the cells along with any markers we wish to analyze. We tested this hypothesis using \gls{colb}, \gls{cold} or \gls{hbss}, and then analyzed the cells via flow cytometry to assess if the enzymes would cleave off markers of interest. The histograms in the \gls{cold} group were similar to that of the buffer group, while the \gls{colb} group visibly lowered CD62L and CD4, indicating partial enzymatic cleavage (\cref{fig:collagenase_fx}). Based on this result, we used \gls{cold} moving forward. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/collagenase.png} \endgroup \caption[Effects of Collagenase Treatment on T cells] {T cells treated with either \gls{colb}, \gls{cold}, or buffer and then stained for various surface markers and analyzed via flow cytometry.} \label{fig:collagenase_fx} \end{figure*} When either adding more \glspl{dms}, removing \glspl{dms} using \gls{cold}, or doing nothing, we observed that, counterintuitively, cell growth seemed to be inhibited in the \textit{added} group while the cells seemed to grow faster in the \textit{removed} group relative to the \textit{no change} group (\cref{fig:add_rem_growth}). Additionally, the \textit{removed} group seemed to have a negative growth rate in the final \SI{4}{\day} of culture, indicating that either the lack activation signal had slowed cell growth or that the cells were growing fast enough to outpace the media feeding schedule. The viability was the same between all groups, indicating that this negative growth rate and the lower growth rate in the \textit{added} group were likely not due to cell death (\cref{fig:add_rem_viability}). Interestingly, the \textit{added} group had significantly higher \pth{} cells compared to the \textit{no change} group, and the inverse was true for the \textit{removed} group (\cref{fig:add_rem_cd4}). These results show that the growth rate and phenotype are fundamentally altered by changing the number of \glspl{dms} temporally. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/add_remove_endpoint.png} \phantomsubcaption\label{fig:add_rem_growth} \phantomsubcaption\label{fig:add_rem_viability} \phantomsubcaption\label{fig:add_rem_cd4} \endgroup \caption[Results of Adding/Removing \acrshort{dms} on Day 4] {Changing \gls{dms} concentration on day 4 has profound effects on phenotype and growth. \subcap{fig:add_rem_growth}{Longitudinal fold change}, \subcap{fig:add_rem_viability}{longitudinal viability}, and \subcap{fig:add_rem_cd4}{day 14 \pthp{}} of T cell cultures with \glspl{dms} added, removed, or kept the same on day 4. } \label{fig:add_rem} \end{figure*} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/spade_gates.png} \endgroup \caption[\acrshort{spade} Gating Strategy] {Gating strategy for quantifying early-differentiated T cells via \gls{spade}.} \label{fig:spade_gates} \end{figure*} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/add_remove_spade.png} \phantomsubcaption\label{fig:spade_msts} \phantomsubcaption\label{fig:spade_quant} \phantomsubcaption\label{fig:spade_tsne_all} \phantomsubcaption\label{fig:spade_tsne_stem} \endgroup \caption[\acrshort{spade} and \acrshort{tsne} Analysis of Temporally Modulated \acrshort{dms} Cultures] {Removing \glspl{dms} leads to a higher fraction of potent stem-memory T cells compared to both adding and not changing the \gls{dms} concentration at day 4. \subcap{fig:spade_msts}{SPADE plots of CD4, CD45RA, CD27, and CD45RO expression on T cells. All cells from the added, removed, or no change groups were pooled and clustered at once.} \subcap{fig:spade_quant}{T cells from SPADE plots clustered by expression in (\subref{fig:spade_msts}) quantified against total cell number from each group.} \subcap{fig:spade_tsne_all}{\gls{tsne} plots of all cells pooled from all groups.} \subcap{fig:spade_tsne_stem}{\gls{tsne} plots of T cells from all groups manually gated on \cdp{8}\cdp{27}\cdp{45RO}.} } \label{fig:spade} \end{figure*} We next asked what the effect of removing the \glspl{dms} would have on other phenotypes, specifically \gls{tcm} and \gls{tscm} cells. To this end we stained cells using a 34-marker mass cytometry panel and analyzed them using a Fluidigm Helios. After pooling the \gls{fcs} file events from each group and analyzing them via \gls{spade} we saw a strong bifurcation of CD4 and CD8 T cells. When looking at CD27, CD45RA, and CD45RO (markers commonly used to identify \gls{tcm} and \gls{tscm} subtypes) we saw clear ``metaclusters'' composed of individual \gls{spade} clusters which are high for these markers (\cref{fig:spade_msts,fig:spade_gates}). We then gated each of these metaclusters according to their marker levels and assigned them to one of three phenotypes for both the CD4 and CD8 compartments: \gls{tcm} (high CD45RO, low CD45RA, high CD27), \gls{tscm} (low CD45RO, high CD45RA, high CD27), and ``transitory'' \gls{tscm} cells (mid CD45RO, mid CD45RA, high CD27). Together these represent low differentiated cells which should be highly potent as anti-tumor therapies. When quantifying the number of cells from each experimental group in these phenotypes, the number of lower differentiated cells was much higher in the \textit{no change} or \textit{removed} groups compared to the \textit{added} group (\cref{fig:spade_quant}). Furthermore, the \textit{removed} group had a much higher fraction of \gls{tscm} cells compared to the \textit{no change} group, which had more ``transitory \gls{tscm} cells.'' The majority of these cells were \cdp{8} cells. When analyzing the same data using \gls{tsne}, we observed a higher fraction of CD27 and lower fraction of CD45RO in the \textit{removed} group (\cref{fig:spade_tsne_all}). Manually gating on the CD27+CD45RO- population more cells with this phenotype in the \textit{removed} group (\cref{fig:spade_tsne_stem}). Together, these data indicate that removing \glspl{dms} at lower timepoints leads to higher expansion, lower \pthp{}, and higher fraction of lower differentiated T cells such as \gls{tscm}, and adding \gls{dms} does the inverse. \subsection{Blocking Integrin Does Not Alter Expansion or Phenotype} One of the reasons the \gls{dms} platform might perform better than the beads is the fact that they are composed of gelatin, which is a collagen derivative. The beads are simply \gls{mab} attached to a polymer resin coated onto an iron oxide core, and thus have no analogue for collagen. Collagen domains present on the \gls{dms} group could provide pro-survival and pro-expansion signals to the T cells through \gls{a2b1} and \gls{a2b2}, causing them to grow better in the \gls{dms} system. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/integrin_1.png} \phantomsubcaption\label{fig:inegrin_1_overview} \phantomsubcaption\label{fig:inegrin_1_fc} \phantomsubcaption\label{fig:inegrin_1_mem} \phantomsubcaption\label{fig:inegrin_1_cd49} \endgroup \caption[Integrin Blocking I] {Blocking with integrin does not lead to differences in memory or growth. \subcap{fig:inegrin_1_overview}{Experimental overview} \subcap{fig:inegrin_1_fc}{Fold change of \gls{dms}-activated T cell over time with each blocking condition.} \subcap{fig:inegrin_1_mem}{\ptmemp{} at day 14 for each blocked condition.} \subcap{fig:inegrin_1_cd49}{\gls{a2b1} and \gls{a2b2} expression over time.} `A' and `B' refer to the inclusion of \anti{\gls{a2b1}} or \anti{\gls{a2b2}} respectively. } \label{fig:integrin_1} \end{figure*} \begin{table}[!ht] \centering \caption{Regression for day 14 phenotype shown in \cref{fig:integrin_1}} \label{tab:integrin_1_reg} \input{../tables/integrin_1_reg.tex} \end{table} We tested this hypothesis by adding blocking \glspl{mab} against \gls{a2b1} and/or \gls{a2b2} to running T cell cultures activated using the \glspl{dms}. These blocking \glspl{mab} were added at day 6 of culture when \gls{a2b1} and \gls{a2b2} were known to be expressed\cite{Hemler1990}. The fold expansion was identical between the blocked and unblocked groupds (\cref{fig:inegrin_1_fc}). Furthermore, the \ptmemp{} (total and across the CD4/CD8 compartments) was not significantly different between any of the groups (\cref{fig:inegrin_1_mem,tab:integrin_1_reg}). Furthermore, \gls{a2b1} and \gls{a2b2} were present on the surface of a significant subset of T cells at day 6, showing that the target we wished to block was present (\cref{fig:inegrin_1_cd49}). \begin{figure*}[ht!] \begingroup \includegraphics{../figures/integrin_2.png} \phantomsubcaption\label{fig:inegrin_2_overview} \phantomsubcaption\label{fig:inegrin_2_fc} \phantomsubcaption\label{fig:inegrin_2_mem} \endgroup \caption[Integrin Blocking II] {Blocking with integrin does not lead to differences in memory or growth. \subcap{fig:inegrin_1_fc}{Fold change of \gls{dms}-activated T cell over time with each blocking condition.} \subcap{fig:inegrin_1_mem}{\ptmemp{} at day 14 for each blocked condition.} `A' and `B' refer to the inclusion of \anti{\gls{a2b1}} or \anti{\gls{a2b2}} respectively. } \label{fig:integrin_2} \end{figure*} \begin{table}[!ht] \centering \caption{Regression for day 14 phenotype shown in \cref{fig:integrin_2}} \label{tab:integrin_2_reg} \input{../tables/integrin_2_reg.tex} \end{table} Since this initial experiment gave a negative result, we decided to block \gls{a2b1} and \gls{a2b2} harder by adding \glspl{mab} at more timepoints between day 0 and day 6, hypothesizing that the majority of the signaling would be during the period of culture where the \gls{dms} surface concentration was at its maximum. Once again, there was no difference between the blocked and unblocked conditions in regard to expansion (\cref{fig:inegrin_2_fc}). Furthermore, none of the \ptmemp{} readouts (total, CD4, or CD8) were statistically different between groups (\cref{fig:inegrin_2_mem,tab:integrin_2_reg}). Taken together, these data suggest that the advantage of the \gls{dms} platform is not due to signaling through \gls{a2b1} or \gls{a2b2}. \subsection{Blocking IL15 Does Not Alter Expansion or Phenotype} \il{15} is a cytokine responsible for memory T cell survival and maintenance. Furthermore, previous experiments showed that it is secreted to a much greater extend in \gls{dms} compared to bead cultures (\cref{fig:doe_luminex}). One of our driving hypotheses in designing the \gls{dms} system was that the higher cell density would lead to greater local signaling. Since we observed higher \ptmemp{} across many conditions, we hypothesized that \il{15} may be responsible for this, and further that the unique \textit{cis/trans} activity of \il{15} may be more active in the \gls{dms} system due to higher cell density. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/il15_blockade_1.png} \phantomsubcaption\label{fig:il15_1_overview} \phantomsubcaption\label{fig:il15_1_fc} \phantomsubcaption\label{fig:il15_1_viability} \phantomsubcaption\label{fig:il15_1_mem} \endgroup \caption[IL15 Blocking I] {Blocking \ilXVra{} does not lead to differences in memory or growth. \subcap{fig:il15_1_overview}{Experimental overview}. Longitudinal measurements of \subcap{fig:il15_1_fc}{fold change} and \subcap{fig:il15_1_viability}{viability} for blocked and unblocked conditions expanded with either beads or \glspl{dms}. \subcap{fig:il15_1_mem}{Flow cytometry markers for \gls{dms}-expanded T cells at day 14 for blocked and unblocked groups.}. } \label{fig:il15_1} \end{figure*} We first tested this hypothesis by blocking \ilXVra{} with either a specific \gls{mab} or an \gls{igg} isotype control at \SI{5}{\ug\per\ml}\cite{MirandaCarus2005}. There was no difference in the expansion rate of blocked or unblocked cells (this experiment also had bead-based groups but they did not expand well and thus were not included) (\cref{fig:il15_1_fc}). Furthermore, there were no differences in viability between any group (\cref{fig:il15_1_viability}). We also performed flow cytometry to asses the \ptmemp{} and \pthp{} outputs. Without even gating the samples, simply lining up their histograms showed no difference between any of the markers, and by extension showing no difference in phenotype (\cref{fig:il15_1_mem}). \begin{figure*}[ht!] \begingroup \includegraphics{../figures/il15_blockade_2.png} \phantomsubcaption\label{fig:il15_2_overview} \phantomsubcaption\label{fig:il15_2_fc} \phantomsubcaption\label{fig:il15_2_viability} \phantomsubcaption\label{fig:il15_2_mem} \endgroup \caption[IL15 Blocking II] {Blocking soluble \il{15} does not lead to differences in memory or growth. \subcap{fig:il15_2_overview}{Experimental overview}. Longitudinal measurements of \subcap{fig:il15_2_fc}{fold change} and \subcap{fig:il15_2_viability}{viability} for blocked and unblocked conditions expanded with \glspl{dms}. \subcap{fig:il15_2_mem}{Flow cytometry markers for \gls{dms}-expanded T cells at day 14 for blocked and unblocked groups.} } \label{fig:il15_2} \end{figure*} We next tried blocking soluble \il{15} itself using either a \gls{mab} or an \gls{igg} isotype control. Anti-\il{15} or \gls{igg} isotype control was added at \SI{5}{\ug\per\ml}, which according to \cref{fig:doe_luminex} was in excess of the \il{15} concentration seen in past experiments by over \num{20000} times. Similarly, there was no difference between fold change, viability, or marker histograms between any of these markers, showing that blocking \il{15} led to no difference in growth or phenotype. In summary, this data did not support the hypothesis that the \gls{dms} platform gains its advantages via the \il{15} pathway. \section{Discussion} This work provides insight for how the \gls{dms} platform operates and how it may be optimized further. The data showing increased \pthp{} when \glspl{dms} are added and the reverse when removed is consistent with other data we produced via \gls{doe} showing that higher \gls{dms} concentrations lead to higher \pthp{} (\cref{fig:doe_responses_cd4,fig:add_rem_cd4}). The difference in this case is that altering activation signal analogously affects the \pthp{} in the dimension of time as well as space. A similar trend was observed with memory T cells in this aim. Our previous \gls{doe} data showed that, to a point, lower \gls{dms} concentration leads to higher \ptmemp{} (\cref{fig:doe_responses_mem}). In this aim, we showed that decreasing activation signal temporally by removing \glspl{dms} leads to the same effect in the \gls{tcm}, \gls{tscm} and ``transitory'' \gls{tscm} populations, (all of which are included in the \ptmem{} phenotype). Taken together, these imply that temporally or spatially decreasing the \gls{dms} concentration, and thus the activation signal, increases memory and lowers CD4+ fractions. While we did not find support for our hypothesis that \glspl{dms} signal via the \gls{a2b1} and/or \gls{a2b2} receptors, we can speculate that either the experiment failed to block the targeted pathways or that this mechanism is simply not relevant for our system. On the first point, we did not verify that these \glspl{mab} actually blocked their target receptors (although they were from a reputable manufacturer, \bl). However, other groups have shown that these particular clones work at the concentrations we used\cite{MirandaCarus2005}. Furthermore, we can safely rule out the possibility that the \glspl{mab} never reached their targets, as they were added immediately after the T cells were resuspended as required for cell counting, hence their resting clustered state was disrupted. Therefore, the most likely failure mode was that the \glspl{mab} we obtained were somehow defective in their intended purpose, which we could experimentally verify using adhesion assays. On the second point, collagen domains may not even be relevant to our system depending on the extent of \gls{stp} coating. We intended by design for the system to be fully coated with \gls{stp} (\cref{fig:stp_coating}). Thus the domains that \gls{a2b1} and \gls{a2b2} may be targeting could be sterically hindered by a layer of \gls{stp}, and if not that, also a layer of CD3/CD28 \glspl{mab}. The other possibility is that these domains are simply denatured to beyond recognition due to the fabrication process for the microcarriers (which involves a proprietary cross-linking step to make the material autoclave-safe). Either of these could be tested and verified by staining the \glspl{dms} with a fluorescently-tagged \gls{mab} and verifying binding via confocal microscopy or indirect protein quantification as we do for the \gls{qc} of the \gls{dms}. If this test came back negative, we would be fairly confident that the \gls{a2b1} and \gls{a2b1} domains are either unreachable or unrecognizable. Even if it turned out that collagen binding domains are non-existent in the \gls{dms} system, previous studies have shown that these domains can enhance proliferation and survival, and thus adding them along with with the \glspl{mab} could enhance T cell expansion\cite{Aoudjit2000, Gendron2003, Boisvert2007}. We also failed to uphold our hypothesis that the \gls{dms} system gains its advantage via \il{15} signaling. There could be multiple reasons for why blocking either \il{15} itself or its receptor would not influence the response at all. First, it could be that \il{15} is not important in our system, which is not likely given the importance of \il{15} in T cells expansion and particularly memory phenotypes\cite{Lodolce1998,Kennedy2000}. Second, in the case of the receptor it could be that that \glspl{mab} we purchased did not actually block, which also seems unlikely given that this clone has been observed to inhibit proliferation in the past (although like the integrin blocking experiments we did not verify for ourselves that it blocked), albeit of resting T cells\cite{MirandaCarus2005}. Third, it could be that turnover of the receptor was so high that there were not enough \glspl{mab} to block (the key difference between our experiment and that of \cite{MirandaCarus2005} was that they used resting T cells, which are not expressing protein to nearly as high of a degree). The way to test this would be to simply titrate increasing concentrations of \gls{mab} (which we did not do in our case because the \gls{mab} was already very expensive in the concentrations employed for our experiment). Fourth, blocking the soluble protein may not have worked because \il{15} may have been secreted and immediately captured via \ilXVra{} either by the cell that secreted it or by a neighboring cell. Regardless of whether or not \il{15} is important for the overall mechanism that differentiates the \glspl{dms} from the beads, adding \il{15} or its receptor complex to the surface of the \gls{dms} might produce interesting and positive results on expansion and memory phenotype. Essentially this would turn the \glspl{dms} into stromal cells that present \il{15}, as seen to be important in the early work with \il{15} in mice\cite{Lodolce1998}. \chapter{AIM 3}\label{aim3} \section{Introduction} % DO NOT COMMENT OUT THIS LINE: the real purpose of this aim was to appease % Nature Biotech because they think that animal models are necessary for good % science. This entire aim exists because of their foolishness. The purpose of this aim was to verify that \gls{car} T cells produced using the \gls{dms} system will show potent anti-tumor properties in a complex \invivo{} system compared to state-of-the-art bead technology\footnote{adapted from \dmspaper{}}. We hypothesized that due to the increased \ptmem{} and \pth{} phenotypes as shown in \cref{aim1}, that \gls{dms}-expanded T cells would show longer survival and lower tumor burden than those expanded with beads. We explored the effect of dosing at different levels and the effect of harvesting T cells at early timepoints in the culture, which has been shown to produce lower-differentiated T cells with higher potency\cite{Ghassemi2018}. \section{Methods} \subsection{T Cell Culture} T cells were grown as described in \cref{sec:tcellculture}. \subsection{CD19-CAR T Cell Generation} T cells were grown as described in \cref{sec:transduction}. \subsection{\Invivo{} Therapeutic Efficacy in NSG Mice Model} % METHOD describe how the luciferase cells were generated (eg the kwong lab) % METHOD use actual product numbers for mice All mice in this study were male \gls{nsg} mice from Jackson Laboratories. At day 0 (\SI{-7}{\day} relative to T cell injection), \num{1e6} firefly luciferase-expressing\footnote{luciferase transduction was performed and verified by Ian Miller in the Kwong Lab at Georgia Tech} \product{\nVI{} cells}{ATCC}{CRL-3273} suspended in ice-cold \gls{pbs} were injected via tail vein into each mouse. At day 7, saline or \gls{car} T cells at the indicated doses from either bead or \gls{dms}-expanded T cell cultures (for \SI{14}{\day}) were injected into each mouse via tail vein. Tumor burden was quantified longitudinally via an \gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug} luciferin at \SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally under isoflurane anesthesia into each mouse and allowed to circulate for at least \SI{10}{\minute} before imaging. Mice were anesthetized again and imaged using the \gls{ivis}. Mice from each treatment group/dose were anesthetized, injected, and imaged together; exposure time of the \gls{ivis} was limited to avoid saturation based on the signal from the saline group. \gls{ivis} images were scaled to common minimum and maximum photon counts. Endpoint for each mouse was determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis, blindness, lethargy, and weight loss). Mice were euthanized according to these endpoint criteria using carbon dioxide asphyxiation. \subsection{Statistics} Survival curves were created and statistically analyzed using GraphPad Prism using the Mantel-Cox test to assess significance between survival groups. \section{Results} \subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/mouse_dosing_overview.png} \endgroup \caption[Mouse Dosing Experimental Overview] {Overview of \invivo{} experiment to test \gls{car} T cells expanded with either \glspl{dms} at different doses. } \label{fig:mouse_dosing_overview} \end{figure*} \begin{table}[!ht] \centering \caption{Cells injected for \acrshort{car} T cell \invivo{} dose study} \label{tab:mouse_dosing_results} \input{../tables/mouse_dose_car.tex} \end{table} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/mouse_dosing_qc.png} \phantomsubcaption\label{fig:mouse_dosing_qc_mem} \phantomsubcaption\label{fig:mouse_dosing_qc_cd4} \phantomsubcaption\label{fig:mouse_dosing_qc_growth} \endgroup \caption[Mouse Dosing T Cell Characteristics] {Characteristics of T cells harvested at day 14 injected into NSG mice at varying doses. Fractions of T cell subtypes in the day 14 product including \subcap{fig:mouse_dosing_qc_mem}{\ptmemp{}}. \subcap{fig:mouse_dosing_qc_cd4}{\pthp{}}, and \subcap{fig:mouse_dosing_qc_growth}{Fold change of T cells.} } \label{fig:mouse_dosing_qc} \end{figure*} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/mouse_dosing_ivis.png} \phantomsubcaption\label{fig:mouse_dosing_ivis_images} \phantomsubcaption\label{fig:mouse_dosing_ivis_plots} \phantomsubcaption\label{fig:mouse_dosing_ivis_survival} \phantomsubcaption\label{fig:mouse_dosing_ivis_survival_comp} \phantomsubcaption\label{fig:mouse_dosing_ivis_survival_full} \endgroup \caption[Mouse Dosing IVIS and Survival Results] {T cells expanded with \glspl{dms} confer greater anti-tumor potency \invivo{} even at lower doses. \subcap{fig:mouse_dosing_ivis_images}{IVIS images of \nVI{} tumor-bearing \gls{nsg} mice injected with varying doses of T cells} \subcap{fig:mouse_dosing_ivis_plots}{Plots showing quantified photon counts of the results from (\subref{fig:mouse_dosing_ivis_plots}).} \subcap{fig:mouse_dosing_ivis_survival}{Survival plots of mice} \subcap{fig:mouse_dosing_ivis_survival_comp}{Survival plots of mice showing only those that received a comparable number of \gls{car} T cells.} \subcap{fig:mouse_dosing_ivis_survival_full}{The same data as \subref{fig:mouse_dosing_ivis_survival} except showing the full time until euthanasia for all mice (including those that died via \gls{gvhd}). Survival curves were statistically analyzed using the Mantel-Cox test in GraphPad Prism.} } \label{fig:mouse_dosing_ivis} \end{figure*} We asked if the higher memory/naive phenotype and more balanced CD4/CD8 ratio of our \gls{dms}-expanded \gls{car} T cells would lead to better anti-tumor potency \invivo{} compared to bead-expanded \gls{car} T cells. We also asked if this superior anti-tumor potency would hold true at lower doses of \gls{car} expressing T cells in the DMS group vs the bead group. To test this, we used a human xenograft model of B cell \gls{all} by intravenously injecting \gls{nsg} mice with \num{1e6} \nVI{} tumor cells expressing firefly luciferase\cite{Fraietta2018}. After \SI{7}{\day} of tumor cell growth (\cref{fig:mouse_dosing_overview}), we intravenously injected saline or three doses (high, medium, and low) of \gls{car} T cells from either bead or \gls{dms} cultures expanded for \SI{14}{\day}. We quantified \ptcarp{} bead and \gls{dms} groups using the \gls{ptnl} assay (\cref{tab:mouse_dosing_results}). Before injecting the T cells into the mice, we quantified their phenotype and growth. We observed that for this expansion, the bead and \gls{dms} T cells produced similar numbers of \ptmem{} T cells, and the beads even had a higher fraction of \cdp{45RA} cells, which is present on lower-differentiated \glspl{tn} and \glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the \pthp{} of the final product was higher in \gls{dms} (\cref{fig:mouse_dosing_qc_cd4}). The \gls{dms} T cells also expanded more robustly than the beads (\cref{fig:mouse_dosing_qc_growth}). In the \nVI{}/\gls{nsg} xenograft model, bead and \gls{dms}-treated mice at all doses had lower tumor burden and significantly longer survival compared to the saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at each dose the \gls{dms}-treated mice had much lower tumor burden and significantly higher survival than their bead-treated counterparts (\cref{fig:mouse_dosing_ivis_survival}). When factoring the percentage T cells in each dose that expressed the \gls{car}, survival of the low \gls{dms} dose (which had similar total \gls{car} T cells compared to the bead medium dose and less than the bead high dose) was significantly higher than that of both the bead medium dose and the bead high dose (\cref{fig:mouse_dosing_ivis_survival_comp}). Overall, the Kaplan-Meier survival of \nVI{} tumor bearing \gls{nsg} mice shown in the \cref{fig:mouse_dosing_ivis_survival} was up to day 40 as reported elsewhere\cite{Fraietta2018}. However, most of the mice euthanized from day 40 through day 46 from \gls{dms} groups showed no or very small fragment of spleen which was due to \gls{gvhd} responses (\cref{fig:mouse_dosing_ivis_survival_full}). Similar \gls{gvhd} responses \SIrange{40}{50}{\day} after injection have been reported by others in \gls{nsg} mice injected with human \gls{pbmc}\cite{Ali2012}. Both survival analyses (up to day 40 in \cref{fig:mouse_dosing_ivis_survival} and up to day 46 in \cref{fig:mouse_dosing_ivis_survival_full}) confirmed that \gls{dms}-expanded groups outperformed bead-expanded groups in terms of prolonging survival of \nVI{} tumor challenged \gls{nsg} mice. Together, these data suggested that \glspl{dms} produce T cells that are not only more potent that bead-expanded T cells (even when accounting for differences in \gls{car} expression) but also showed that \gls{dms} expanded T cells are effective at lower doses. Given the \gls{qc} data of T cells prior to injection, it seems that this advantage for \gls{dms} groups was either due to higher \pthp{} or greater overall fitness (implied by higher fold change) (\cref{fig:mouse_dosing_qc_cd4,fig:mouse_dosing_qc_growth}). It was likely not due to memory phenotype given that this was actually slightly higher for the bead culture (\cref{fig:mouse_dosing_qc_mem}). \subsection{Beads and DMSs Perform Similarly at Earlier Timepoints} We then asked how T cells activated using beads or \gls{dms} performed when harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same experiments as described in \cref{fig:mouse_dosing_overview} with the modification that T cells were only expanded and harvested after \SI{6}{\day}, \SI{10}{\day}, or \SI{14}{\day} of expansion (\cref{fig:mouse_timecourse_overview}). T cells were frozen after harvest, and all timepoints were thawed simultaneously prior to injection. The dose of T cells injected was \num{1.25e6} cells per mouse (the same as the high dose in the first experiment). All other characteristics of the experiment were the same. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/mouse_timecourse_overview.png} \endgroup \caption[Mouse Timecourse Experimental Overview] {Overview of \invivo{} experiment to test \gls{car} T cells using either \glspl{dms} or bead harvested at varying timepoints. } \label{fig:mouse_timecourse_overview} \end{figure*} As was the case with the first \invivo{} experiment, \gls{dms} cultures expanded much more efficiently than bead cultures (\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} at each timepoint, the bead group had much higher \ptcar{} expression at earlier timpoints compared to \gls{dms}, while they equalized at later timepoints (\cref{fig:mouse_timecourse_qc_car}). In addition, overall \ptcar{} expression decreased at later timepoints, indicating that transduced cells either grew slower or died faster compared to untransduced cells. The \pthp{} was higher overall in \gls{dms} groups but decreased with increasing timepoints (\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6 between bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{} at day 14 despite the overall \ptmemp{} decreasing with time (\cref{fig:mouse_timecourse_qc_mem}). \begin{figure*}[ht!] \begingroup \includegraphics{../figures/mouse_timecourse_qc.png} \phantomsubcaption\label{fig:mouse_timecourse_qc_growth} \phantomsubcaption\label{fig:mouse_timecourse_qc_car} \phantomsubcaption\label{fig:mouse_timecourse_qc_cd4} \phantomsubcaption\label{fig:mouse_timecourse_qc_mem} \endgroup \caption[Mouse Timecourse T Cell Characteristics] {Characteristics of T cells harvested at varying timepoints injected into NSG mice. \subcap{fig:mouse_timecourse_qc_growth}{Fold change of T cells (each timepoint only includes the runs that were harvested at day 14).} Fractions of T cell subtypes in the day 14 product including \subcap{fig:mouse_timecourse_qc_car}{\ptcarp{}}, \subcap{fig:mouse_timecourse_qc_cd4}{\pthp{}}, and \subcap{fig:mouse_timecourse_qc_mem}{\ptmemp{}}. } \label{fig:mouse_timecourse_qc} \end{figure*} Analyzing the tumor burden using \gls{ivis} showed that mice who received T cells from any group had less tumor than those that received only saline (\cref{fig:mouse_timecourse_ivis}). Unlike the previous experiment, most mice survived until day 40 after which \gls{gvhd} began to take effect (upon euthanization at day 42, most had little or no spleen). When comparing bead and \gls{dms} groups, the \gls{dms} groups had lower tumor than the bead group, at least initially (note that in this experiment they had similar numbers of \ptcar{} cells). For day 6 groups, both treatments seemed to eradicate the tumor initially, then it came back after \SI{21}{\day} for the beads and \SI{28}{\day} for \glspl{dms}. The day 10 groups performed somewhere in between, where they increased linearly unlike the day 6 groups but not as quickly as the day 14 groups. In the case of the \gls{dms} day 10 group, a few mice actually had less tumor burden overall than all other groups. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/mouse_timecourse_ivis.png} \phantomsubcaption\label{fig:mouse_timecourse_ivis_images} \phantomsubcaption\label{fig:mouse_timecourse_ivis_plots} \endgroup \caption[Mouse Timecourse IVIS Results] {\glspl{dms} exhibit superior anti-tumor activity \invivo{} at day 14 compared to beads but are similar to beads at lower timepoints. \subcap{fig:mouse_timecourse_ivis_images}{IVIS images for day 6 to day 42 of mice treated with varying doses of \gls{car} T cells grown with beads or \glspl{dms}.} \subcap{fig:mouse_timecourse_ivis_plots}{Quantified dotplots of the images in (\subref{fig:mouse_timecourse_ivis_images}). Numbers beneath each dot represent the number of mice at that timepoint.}, } \label{fig:mouse_timecourse_ivis} \end{figure*} \section{Discussion} \begin{figure*}[ht!] \begingroup \includegraphics{../figures/mouse_summary.png} \phantomsubcaption\label{fig:mouse_summary_1} \phantomsubcaption\label{fig:mouse_summary_2} \endgroup \caption[Mouse Summary] {Summary of T cells injected into mice for the \subcap{fig:mouse_summary_1}{first} and \subcap{fig:mouse_summary_2}{second} experiments. The y-axis maximum is set to the maximum cell number injected between both experiments (\num{1.25e6}). NOTE: the \gls{car} was quantified using a separate panel from the other markers. } \label{fig:mouse_summary} \end{figure*} When we tested bead- and \gls{dms}-expanded \gls{car} T cells, the latter prolonged survival compared to the former in \nVI{} tumor challenged (intravenously injected) \gls{nsg} mice. This held true when matching groups for absolute \gls{car} dose. Furthermore, \gls{dms}-expanded \gls{car} T cells were effective in clearing tumor cells as early as \SI{7}{\day} post T injection even at low dose compared to the bead groups where tumor burden was higher than \gls{dms} groups across all the total T cell doses tested here. These suggest that \glspl{dms} (compared to beads) produced highly effective \gls{car} T cells that can efficiently kill tumor cells. When comparing total number of injected T cells with different phenotypes, the number of \ptmem{} (both with and without CD45RA) cells was lower in the low-dose \gls{dms} group compared to the med-dose bead group (which had similar numbers of \gls{car} T cells) (\cref{fig:mouse_summary_1}). This could mean several things. First, the \ptmem{} phenotype may have nothing to do with the results seen here, at least in this model. While this may have been the case in our hands, this would contradict previous evidence suggesting that \gls{tn} and \gls{tcm} cells work better in almost the same model (the only difference being Raji cells in place of \nVI{} cells, both of which express CD19)\cite{Sommermeyer2015}. Second, the distribution of \gls{car} T cells across different subtypes of T cells was different between the \gls{dms} and bead groups (with possibly higher correlation of \gls{car} expression and the \ptmem{} phenotype). It is hard to assess this without strong assumptions as the \gls{car} was quantified using a separate flow panel relative to the other markers. We can make a similar observation for the number of \pth{} T cells injected (\cref{fig:mouse_summary_1}). In this case, either the \pth{} phenotype doesn't matter in this model (or the \ptk{} population matters much more), or the distribution of \gls{car} is different between CD4 and CD8 T cells in a manner that favors the \gls{dms} group. Previous groups have shown that \pthp{} T cells are important for response (albeit for a glioblastoma model and not a B-cell \gls{all} model)\cite{Wang2018}. When testing \gls{car} T cells at earlier timepoints relative to day 14 as used in the first \invivo{} experiment, none of the \gls{car} treatments seemed to work as well as they did in the first experiment. However, the total number of \gls{car} T cells was generally much lower in this second experiment relative to the first (\cref{fig:mouse_summary}). Only the day 6 group had \gls{car} T cell numbers comparable to the weakest dose of bead cells given in the first experiment, and these T cells were harvested at earlier timepoints than the first mouse experiment and thus are not directly comparable. Furthermore, the \ptcarp{} decreased over time, which suggested that the transduced T cells grew slower. This has been observed elsewhere and could be due to tonic signaling\cite{GomesSilva2017}. The lower overall \gls{car} doses may explain why at best, the tumor seemed to be in remission only temporarily. Even so, the \gls{dms} group seemed to perform better at day 6 as it held off the tumor longer, and also slowed the tumor progression relative to the bead group at day 14 (\cref{fig:mouse_timecourse_ivis_plots}). Taken together, these data suggest that the \gls{dms} platform produces T cells that have an advantage \invivo{} over beads. While we may not know the exact mechanism, our data suggests that the responses are unsurprisingly influenced by the \ptcarp{} of the final product. Followup experiments are needed to determine the precise phenotype responsible for these results. \chapter{CONCLUSIONS AND FUTURE WORK}\label{conclusions} \section{Conclusions} This dissertation describes the development of a novel T cell expansion platform, including the fabrication, \gls{qc}, and biological validation of its performance both \invitro{} and \invivo{}. Development of such a system would have been meaningful even if it only performed as well as current technology, as adding another method to the arsenal of the growing T cell manufacturing industry would reduce the reliance on a small number of companies that currently license magnetic bead-based T cell expansion reagents. However, we additionally demonstrated that the \gls{dms} platform expands more T cells on average, including highly potent \ptmem{} and \pth{} T cells, and produces higher percentages of both. If commercialized, this would be a compelling asset the T cell manufacturing industry. In \cref{aim1}, we developed the \gls{dms} platform and verified its efficacy \invitro{}. Importantly, this included \gls{qc} at every critical step of the fabrication process to ensure that the \glspl{dms} can be made within a targeted specification. These \gls{qc} steps all rely on common, cost-effective, easy-to-use assays such as the \gls{haba} assay, \gls{bca} assay, and \gls{elisa}. The microcarriers themselves are an off-the-shelf product available from reputable vendors, and they have a regulatory history in human cell therapies that will aid in clinical translation\cite{purcellmain}. On average, we demonstrated that the \glspl{dms} outperforms bead-based technology in terms of total fold expansion, \ptmemp{}, and \pthp{} by \SI{131}{\percent}, \SI{3.5}{\percent}, and \SI{7.4}{\percent} controlling for donor, operator, and a variety of process conditions. In addition to larger numbers of potent T cells, other advantages of our approach are that the \glspl{dms} are large enough to be filtered (approximately \SI{300}{\um}) using standard \SI{40}{\um} cell strainers or similar. If the remaining cells inside that \glspl{dms} are also desired, digestion with dispase or collagenase may be used. \gls{cold} may be selective enough to dissolve the \gls{dms} yet preserve surface markers which may be important to measure as critical quality attributes \glspl{cqa} (\cref{fig:collagenase_fx}). Furthermore, our system should be compatible with large-scale static culture systems such as the \gls{grex} bioreactor or perfusion culture systems, which have been previously shown to work well for T cell expansion\cite{Forget2014, Gerdemann2011, Jin2012}. In \cref{aim2a}, we developed a modeling pipeline that can be used by commercial entities to identify \glspl{cqa} and \gls{cpp} during scale-up. These are highly important for a variety of reasons. First, understanding pertinent \glspl{cpp} allow manufacturers to operate their process at optimal conditions. This is important for anti-tumor cell therapies, where the prospects of a patient can urgently depend on receiving therapy in a timely manner. Optimal process conditions allow T cells to be expanded as quickly as possible for the patient, while also minimizing cost for the manufacturer. Second, \glspl{cqa} can be used to define process control schemes as well as release criteria. Process control, and with it the ability to predict future outcomes based on data obtained at the present, is highly important for cell therapies given that batch failures are extremely expensive\cite{Harrison2019}, and predicting a batch failure would allow manufacturers to restart the batch in a timely manner without wasting resources. Furthermore, \glspl{cqa} can be used to define what a ``good'' vs ``bad'' product is, from which dosing and followup procedures in the clinic can be planned more accurately. In the aim, we cannot claim to have found the universal set of \glspl{cqa} and \glspl{cpp}, as we used tissue culture plates instead of a bioreactor and we only used one donor. However, we have indeed outlined a method that others may use to find \glspl{cqa} and \glspl{cpp} for their process. In particular, the 2-phase modeling approach we used (starting with a \gls{doe} and collecting data longitudinally) is a strategy that manufacturers can easily implement. Also, collecting secretome and metabolome is generalizable to most bioreactors and expansion systems, as they can be obtained with relatively inexpensive equipment (Luminex assay, benchtop \gls{nmr}, etc) without disturbing the cell culture. In \cref{aim2b}, we further explored additional tuning knobs that could be used to control and optimize the \gls{dms} system. We determined that altering the \gls{dms} concentration temporally has profound effects on the phenotype and expansion rate. This agrees with other data we obtained in \cref{aim2a} and with what others have generally reported about signal strength and T cell differentiation\cite{Gattinoni2012, Lozza2008, Lanzavecchia2005, Corse2011}. We did not find any mechanistic relationship between either integrin signaling or \il{15} signaling. In the case of the former, it may be more likely that the \glspl{dms} surfaces are saturated to the point of sterically hindering any integrin interactions with the collagen surface. In the case of \il{15}, more experiments likely need to be done in order to plausibly rule out this mechanism and/or determine if it is involved at all. In \cref{aim3} we determined that \gls{dms}-expanded T cells that also performed better than beads \invivo{}. In the first experiment we performed, the results were clearly in favor of the \glspl{dms}. In the second experiment, even the \gls{dms}-expanded cells failed to fully control the tumor burden, but this is not surprising given the low \ptcarp{} across all groups. Also, despite this, the \gls{dms} group appeared to control the tumor better on average for early, mid, and late T cell harvesting timepoints. It was not clear if this effect was due to increased \pthp{}, \ptmemp{}, or fitness of the \gls{dms}-expanded T cells given their higher expansion rate. More data is needed to establish which phenotype is responsible for the results we observed. We did not include the \gls{car} in the same panel as the other phenotype surface markers, making it difficult to reliably assess the identity of the \ptcar{} cells. Finally, while we have demonstrated the \gls{dms} system in the context of \gls{car} T cells, this method can theoretically be applied to any T cell immunotherapy which responds to \acd{3}/\acd{28} \gls{mab} and cytokine stimulation. These include \glspl{til}, virus-specific T cells, T cells engineered to express $\upgamma\updelta$ \glspl{tcr}, $\upgamma\updelta$ T cells, T cells with transduced-\gls{tcr}, and \gls{car}-\gls{tcr} T cells\cite{Cho2015, Straetemans2018, Robbins2011, Brimnes2012, Baldan2015, Walseng2017}. Similar to \glspl{car} against CD19 used in liquid tumors, these T cell immunotherapies would similarly benefit from the increased proliferative capacity, metabolic fitness, migration, and engraftment potential characteristic of naïve and memory phenotypes\cite{Blanc2018, Lalor2016, Rosato2019}. Indeed, since these T cell immunotherapies are activated and expanded with either soluble \glspl{mab} or bead-immobilized \glspl{mab}, our system will likely serve as a drop-in substitution to provide these benefits. \section{Future Directions} There are several important next steps to perform with this work, many of which will be relevent to using this technology in a clinical trial: \subsection{Using GMP Materials} While this work was done with translatability and \gls{qc} in mind, \gls{gmp} are still absent from the fabrication process. The microcarriers themselves are made from porcine-derived collagen, which itself is not \gls{gmp}-compliant due to its non-human animal origins. However, using any other source of collagen should work so long as the structure of the microcarriers remains relatively similar and it has lysine groups that can react with the \gls{snb} to attach \gls{stp} and \glspl{mab}. Obviously these would need to be tested and verified, but these should not be insurmountable. Furthermore, the \gls{mab} binding step requires \gls{bsa} to prevent adsorption to the non-polar polymer walls of the reaction tubes. A human carrier protein such as \gls{hsa} could be used in its place to eliminate the non-human animal origin material, but this could be much more expensive. Alternatively, the use of protein could be replaced altogether by a non-ionic detergent such as Tween-20 or Tween-80, which are already used for commercial \gls{mab} formulations for precisely this purpose\cite{Kerwin2008}. Validating the process with Tween would be the best next step to eliminate \gls{bsa} from the process. The \gls{stp} and \glspl{mab} in this work were not \gls{gmp}-grade; however, they are commonly used in clinical technology such as dynabeads and thus the research-grade proteins used here could be easily replaced. The \gls{snb} is a synthetic small molecule and thus does not have any animal-origin concerns. \subsection{Mechanistic Investigation} Despite the improved outcomes in terms of expansion and phenotype relative to beads, we don't have a good understanding of why the \gls{dms} platform works as well as it does. The following are several plausible hypotheses and testing strategies: \subsubsection{Cytokine Cross-talk} As hypothesized in the beginning of this work, the \gls{dms} may derive their advantage through increased cytokine cross-talk. While this work found that blocking \il{15} did not lead to differences in outcome, other cytokines could be explored in a similar vein. An efficient test of this hypothesis would be to simply incubate T cells grown with either bead or \glspl{dms} with a cocktail of \glspl{mab} each feeding cycle that target the cytokines seen in \cref{fig:doe_luminex}, assuming that at least a few of the targeted cytokines will cause a difference. The experiment should be sized appropriately such that the second order interaction effect can be resolved (that is, the effect of adding the cocktail conditional on the activation method). In these terms, we hypothesize that the growth and phenotype will be more similar between the beads and \glspl{dms} when the cocktail is added, while the \gls{dms} will have better expansion and phenotype when the cocktail is not added. If this experiment shows any effects, the cytokines responsible can be resolved by testing individually (or in small pools). One caveat with this approach is that it assumes that each \gls{mab} in the cocktail is in sufficient quantity to quench their target cytokine between each feed cycle. This assumption can be tested by running Luminex with each cocktail addition. If a given cytokine is undetectable, this indicates that the blocking \gls{mab} completely quenched all target cytokine at the time of addition and in the time between feeding cycles. \subsubsection{Interior Cell Phenotype} Unlike the beads, the \glspl{dms} have interior and exterior surfaces. We demonstrated that some T cell expand on the interior of the \glspl{dms}, and these cells may be phenotypically different than those growing on the exterior. This could lead to an asymmetric cytokine cross-talk which accounts for the population-level differences seen in comparison to the beads. Experimentally, the first step involves separating the \glspl{dms} from the loosely or non-adhered T cells and digesting the \glspl{dms} with \gls{cold} (concentrations of \SI{10}{\ug\per\ml} will completely the \glspl{dms} within \SIrange{30}{45}{\min}) to isolate the interior T cells. Unfortunately, only \SIrange{10}{20}{\percent} of all cells will be on the interior, so this population may only have cells on the order of \num{1e3} to \num{1e4} for analysis. A good first pass experiment would be to analyze both populations with flow cytometry (since flow cytometry is relatively cheap and doesn't require a large number of cells) to simply establish if the two groups are different phenotypes or are in a different state of activation. From there, more in-depth analysis using \gls{cytof} or another high-dimensionality method may be used to evaluate differential cytokine expression. \subsubsection{Antibody Surface Density} While our \gls{doe} experiments showed a relationship between activating \gls{mab} density and number of cells, we don't know how the \gls{dms} \gls{mab} surface density compares to that of the beads. The \gls{mab} surface density on the \glspl{dms} is likely lower given the number of total binding sites on \gls{stp} and the number of \glspl{mab} that actually bind, which may lead to differences in performance\cite{Lozza2008}. Before attempting this experiment, it will be vital to improve the \gls{dms} manufacturing process such that \gls{mab} binding is predictable and reproducible (see below). Once this is established, we can then determine the amount of \glspl{mab} that bind to the beads, which could be quantified much like the \gls{mab} binding step in the \gls{dms} process (eg with ELISA, \cref{fig:dms_flowchart}). Knowing this, we can vary the \gls{mab} surface density for both the bead and the \glspl{dms} using a dummy \gls{mab} as done previously with the \gls{doe} experiments in \cref{aim2a}. Using varying surface densities that are matched per-area between the beads and \glspl{dms} we can then activate T cells and assess their growth/phenotype as a function of surface density and the presentation method. \subsection{Reducing Ligand Variance} While we have robust \gls{qc} for each step of the \gls{dms} coating process, we still see high variance across time and personnel (\cref{fig:dms_coating}). This is less than ideal for translation. The following are a list of variance sources and potential mitigation strategies: \subsubsection{Mass loss during autoclaving} In order to ensure a consistent reaction volume, we mass the tube after adding carriers and \gls{pbs} prior to autoclaving. Autoclaving and washing will cause variations in the liquid level, and these are corrected using the pre-recorded tube mass. However, this assumes that the mass of the tube never changes, which may or may not be true in an autoclave where the temperature easily causes deformation of the plastic tube material. This can easily be tested by autoclaving empty tubes and observing a mass change. If there is a mass change, it may be mitigated by pre-autoclaving (assuming that autoclaving is idempotent with respect to mass loss), or by statistically estimating the bias by recording the mean mass loss for a set of tubes and using this as a correction factor. \subsubsection{Errors in initial microcarrier massing} The massing of microcarriers at the very beginning of the process requires care due to the low target mass and the propensity for both the plastic tubes and microcarriers to accumulate static. Oddly, the biotin attachment readout does not seem to be much affected by the mass of carriers (\cref{fig:dms_qc_doe}); however, this merely means that errors in carrier mass lead to different biotin surface densities, which downstream causes different ratios of \gls{stp} and \gls{mab} attachment since these relationships are non-linear with respect to biotin surface density (\cref{fig:stp_coating,fig:mab_coating}) (this is in addition to the fact that having more or less carriers will bias the total amount of \gls{stp} and \gls{mab} able to bind). A quick survey showed that operators had acceptable margins for error from \SIrange{2.5}{5.0}{\percent} (eg, a target value $X$ \si{\mg} will be accepted as $X$ at plus or minus these margins). These could easily be reduced and standardized via protocol. Additionally, we do not currently record the exact mass of microcarriers weighed for each batch. Knowing this would allow us to pinpoint how much of this variance is due to our acceptable measurement margins and what errors may arise from static and other instrument noise. \subsubsection{Centrifugation after washing} After coating the \glspl{dms} with \gls{snb}, \gls{stp}, or \glspl{mab}, they must be washed. After washing, they must be massed in order to ensure the reaction volume is consistent. Ideally, the tubes are centrifuged after washing to ensure that all liquid is at the bottom prior to beginning the next coating step. Upon survey, not all operators do this, and the protocol is not written to make this obvious. This protocol can be revised followed by additional training. \subsubsection{Accidental microcarrier removal} When washing the microcarriers after a coating step, liquid is aspirated using a stripette. The carriers should be at the bottom of the tube during this aspiration step. Depending on the skill and care of the operator, carriers may be aspirated with the liquid during this step. If this happens, downstream \gls{qc} assays will not reflect the true binding magnitude, as these assays assume the number of carriers is constant. Equipment can be modified (such as aspirators with guides to ensure fixed depth of suction) to mitigate this issue. \subsubsection{BSA binding kinetics} Prior to \gls{mab} addition, \gls{bsa} is added to the reaction volume to block binding to the tubes. \glspl{mab} are added immediately after adding the \gls{bsa}, which means the \gls{bsa} has almost no time to mix completely and thus the \gls{mab} could come into contact with the sides of the tube without competition. This could cause the \gls{mab} \gls{elisa} reading to be lower. This problem may be minor since significant binding would only occur if the \gls{mab}/plastic adhesion was fast and happened in the seconds prior to beginning agitation. We can mitigate this by agitating the tubes with \gls{bsa} for several minutes prior to adding \gls{mab} to ensure mixing. \subsubsection{Improving protein detection} While the \gls{bca} assay and \gls{elisa} are relatively precise, they both have problems that could lead to systemic bias or excess random noise. The \gls{bca} assay is non-specific. All our data shows consistent small (\SI{0.5}{\ug}) but negative readings for blank carriers, which indicates that some background protein (or something that behaves like a protein) may be present that the \gls{bca} assay is detecting. The \gls{elisa} is specific to \glspl{mab}; however, in our case we need to run a blank (just \gls{pbs}, \gls{bsa}, and \glspl{mab} without carriers) and subtract this from the reading, effectively doubling the assay variance. Using \gls{hplc} would mitigate both issues. \gls{hplc} can specifically detect species based on differences in charge and size, so it should be able to quantify \gls{stp} without the extraneous bias of the \gls{bca} assay. In the case of \gls{elisa} it will not remove the need to run a blank, but it should lower variance due to its automated nature. \subsection{Surface Stiffness} The beads and \glspl{dms} are composed of different materials: iron/polymer for the former and cross-linked gelatin for the latter. These materials likely have different stiffnesses, and stiffness could play a role in T cell activation\cite{Lambert2017}. This hypotheses will be difficult to test directly, so it is advised to eliminate other hypothesis before proceeding here. Direct testing could be performed using a force probe to determine the Young's modulus of each material\cite{Ju2017}. Since the microcarriers are porous and the cells will be interacting with the bulk material itself, the void fraction and pore size will need to be taken into account to find the bulk material properties of the cross-linked gelatin\cite{Wang1984}. \subsection{Additional Ligands and Signals on the DMSs} In this work we only explored the use of \acd{3} and \acd{28} \glspl{mab} coated on the surface of the \glspl{dms}. The chemistry used for the \glspl{dms} is very general, and any molecule or protein that could be engineered with a biotin ligand could be attached without any further modification. There are many other ligands (in addition to integrin-binding domains and \il{15} complexes as described at the end of \cref{aim2b}) that could have profound effects on the expansion and quality of T cells which may be utilized. The simplest next step is to simply vary the ratio of \acd{3} and \acd{28} signal. Another obvious example is to attach \il{15}/\ilXVra{} complexes to the surface to mimic \textit{trans} presentation from other cell types\cite{Stonier2010}. Other adhesion ligands or peptides such as GFOGER could be used to stimulate T cells and provide more motility on the \glspl{dms}\cite{Stephan2014}. Finally, viral delivery systems could theoretically be attached to the \gls{dms}, greatly simplifying transduction. \subsection{Assessing Performance Using Unhealthy Donors} All the work presented in this dissertation was performed using healthy donors. This was mostly due to the fact that it was much easier to obtain healthy donor cells and was much easier to control. However, it is indisputable that the most relevant test cases of the \glspl{dms} will be for unhealthy patient T cells, at least for autologous therapies. In particular, it will be interesting to see how the \gls{dms} performs when assessed head-to-head with bead-based expansion technology given that even in healthy donors, the \gls{dms} platform worked where the beads failed (\cref{fig:dms_exp_fold_change}). \subsection{Translation to Bioreactors} In this work we performed some preliminary experiments demonstrating that the \gls{dms} platform can work in a \gls{grex} bioreactor. While an important first step, more work needs to be done to optimize how the \gls{dms} system will or can function in a scalable environment using bioreactors. There are several paths to explore. Firstly, the \gls{grex} itself has additional automation accessories which could be tested, which would allow continuous media exchange and cytokine administration. While this is an improvement from the work done here, it is still a \gls{grex} and has all the disadvantages of an open system. Secondly, other static bioreactors such as the Quantum hollow fiber bioreactor (Terumo) could be explored. Essentially the \gls{dms} would be an additional matrix that could be supplied to this system which would enhance its compatibility with T cells. Finally, suspension bioreactors such as the classic \gls{cstr} or WAVE bioreactors could be tried. The caveat with these is that the T cells only seem to be loosely attached to the \gls{dms} throughout culture, so an initial activation/transduction step in static culture might be necessary before moving to a suspension system (alternatively the \gls{dms} could be coated with additional adhesion ligands to make the T cells attach more strongly). \onecolumn \clearpage \appendix \chapter{META ANALYSIS DATABASE CODE}\label{sec:appendix_meta} The code used to aggregate all experimental data was written primarily in Python, with a subprocess running R in a Docker container to handle the flow cytometry data (\cref{fig:meta_overview}). The PostgreSQL database itself was hosted using \gls{aws} using their proprietary Aurora implementation. \begin{figure*}[ht!] \begingroup \includegraphics{../figures/metaanalysis.png} \endgroup \caption[Meta-analysis Overview] {Overview of strategy used for meta-analysis. Colors: notebook (pink), input files (green), analysis framework (blue), data store (cyan), analysis pipeline (orange).} \label{fig:meta_overview} \end{figure*} The code is available here: \url{https://github.gatech.edu/ndwarshuis3/mdma}. \chapter{META ANALYSIS DONORS}\label{sec:appendix_donors} \begin{table}[!ht] \caption{Donors used in meta-analysis} \begin{subtable}[t]{\textwidth} \centering \caption{characteristics} \input{../tables/donor_chars.tex} \phantomsubcaption\label{tab:meta_donors_chars} \end{subtable} \bigskip \begin{subtable}[t]{\textwidth} \centering \caption{phenotype (all in percents)} \input{../tables/donor_phenotypes.tex} \phantomsubcaption\label{tab:meta_donors_phenotypes} \end{subtable} \label{tab:meta_donors} \end{table} \chapter{BINDING KINETICS}\label{sec:appendix_binding} The binding kinetics of \gls{stp} or \glspl{mab} were simulated using a receptor/ligand model, where the free-floating species in question was the ligand which bound to receptors attached to the microcarriers. Each microcarrier was assumed to be a porous sphere with a fixed number of receptors uniformly distributed throughout its interior matrix. The receptor/ligand reaction was assumed to be instantaneous (which is reasonable given that these are reactions between biotin and \gls{stp} which are extremely strong). From this, we further assumed a spherical interface aligned at the center within each microcarrier wherein all receptors in the interior were unbound and all on the exterior were bound. At $\gls{sym:time}=0$ this interface was assumed to start with a radius equal to that of the microcarrier, and shrunk down to radius of zero as ligand flowed into the porous microcarrier and bound. We assumed the concentration of ligand to be zero at the interface and equal to the bulk concentration at the exterior surface of the microcarrier. Furthermore, we assumed that the interface moved slowly relative to the diffusion of ligand into the microcarriers, and thus we used a quasi-steady-state model to avoid solving a boundary value problem with two movable boundaries (the interface radius and the concentration in bulk). The concentration profile of ligand in the microcarriers is given by Fick's Second Law in spherical coordinates assuming only radial flux and steady state. This with the boundary conditions as stated is: \begin{equation} \label{eqn:binding_ficks} 0 = \frac{1}{\gls{sym:rad}^2} \frac{d}{d\gls{sym:rad}} \left( \gls{sym:rad}^2 \frac{d\gls{sym:mcligconc}}{d\gls{sym:rad}} \right) \end{equation} \begin{equation} \label{eqn:binding_bc_left} \evalat{\gls{sym:mcligconc}}{\gls{sym:rad}=\gls{sym:interrad}} = 0 \end{equation} \begin{equation} \label{eqn:binding_bc_right} \evalat{\gls{sym:mcligconc}}{\gls{sym:rad}=\gls{sym:mcrad}} = \gls{sym:bulkligconc} \end{equation} Solving \cref{eqn:binding_ficks} we find the a relation for the concentration profile in terms of the interfacial radius: \begin{equation} \label{eqn:binding_conc} \gls{sym:mcligconc} = \frac{\gls{sym:bulkligconc}} {(1 / \gls{sym:interrad} - 1 / \gls{sym:mcrad})} \left( \frac{1}{\gls{sym:interrad}} - \frac{1}{\gls{sym:rad}} \right) \end{equation} Solving \cref{eqn:binding_conc} for flux, the molar flow rate into the microcarriers is given by: \begin{equation} \label{eqn:binding_molar_flow} \gls{sym:flowrate} = 4 \pi \gls{sym:mcrad}^2 \evalat{\gls{sym:mcflux}}{\gls{sym:rad} = \gls{sym:mcrad}} = \frac{-4 \pi \gls{sym:appdiff} \gls{sym:bulkligconc}} {1 / \gls{sym:interrad} - 1 / \gls{sym:mcrad}} \end{equation} Using the quasi-steady-state assumption, we can now find time-dependent equations for the interfatial radius and the bulk concentration. The interfacial volume in terms of molar flow rate is given by: \begin{equation} \label{eqn:binding_volume_change} \evalat{\gls{sym:mcrecconc}}{\gls{sym:time} = 0} \frac{d\gls{sym:intervol}}{d\gls{sym:time}} = -\gls{sym:flowrate} \end{equation} Substituting volume of a sphere and applying the chain rule: \begin{equation} \label{eqn:radial_radial_change} \frac{d\gls{sym:interrad}}{d\gls{sym:time}} = \frac{-\gls{sym:flowrate}} {4 \pi \gls{sym:interrad}^2 \evalat{\gls{sym:mcrecconc}}{\gls{sym:time} = 0}} \end{equation} The change in bulk concentration is simply given by: \begin{equation} \label{eqn:radial_conc_change} \frac{d\gls{sym:bulkligconc}}{d\gls{sym:time}} = \frac{-\gls{sym:mcnum}\gls{sym:flowrate}}{\gls{sym:vol}} \end{equation} Substituting \cref{eqn:binding_molar_flow} into \cref{eqn:radial_radial_change} and \cref{eqn:radial_conc_change} yields \cref{eqn:stp_diffusion_1} and \cref{eqn:stp_diffusion_2}. The \gls{stp} binding kinetic profile was fit and calculated using the following MATLAB code. Note that the \inlinecode{geometry} parameter was varied to minimize the \inlinecode{SSE} output. \lstinputlisting{../code/diffusion_stp.m} The geometric diffusivity from above (the \inlinecode{geometry} variable) was used in the below code to obtain the reaction profile for the \gls{mab} binding step. The model is the same except for the parameters which were changed to reflect the \gls{mab} coating process. \lstinputlisting{../code/diffusion_mab.m} \chapter{WASHING KINETICS CODE}\label{sec:appendix_washing} The wash steps for the \glspl{dms} were modeled using the following code: \lstinputlisting{../code/microcarrier_diffusion_washing.m} Complete output from this code is shown below: \input{../code/washing_out.tex} \chapter{REFERENCES} \renewcommand{\chapter}[2]{} % noop the original bib section header \bibliography{references} \bibliographystyle{naturemag} \end{document}