ADD methods for aim 1

tex/thesis.tex

@@ -29,17 +29,11 @@
 
 \setlist[description]{font=$\bullet$~\textbf\normalfont}
 
-\sisetup{per-mode=symbol,list-units=single}
+%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
-\DeclareSIUnit\activityunit{U}
+% acronyms for the lazy
-\DeclareSIUnit\carrier{carriers}
-\DeclareSIUnit\cell{cells}
-\DeclareSIUnit\ab{mAbs}
-\DeclareSIUnit\molar{M}
-\DeclareSIUnit\gforce{\times{} g}
 
-% add acronyms here
 \renewcommand{\glossarysection}[2][]{} % remove glossary title
-\makeglossaries
+\makeglossaries{}
 \newacronym{act}{ACT}{adoptive cell therapies}
 \newacronym{car}{CAR}{chimeric antigen receptor}
 \newacronym[longplural={monoclonal antibodies}]{mab}{mAb}{monoclonal antibody}
@@ -54,13 +48,43 @@
 \newacronym{pdms}{PDMS}{polydimethylsiloxane}
 \newacronym{dc}{DC}{dendritic cell}
 \newacronym{il2}{IL2}{interleukin 2}
+\newacronym{rhil2}{rhIL2}{recombinant human interleukin 2}
 \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{stp}{STP}{streptavidin}
+\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{crispr}{CRISPR}{clustered regularly interspaced short
+  palindromic repeats}
 
 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
-% my commands
+% 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\dms{DMS}
+\DeclareSIUnit\cell{cells}
+\DeclareSIUnit\ab{mAb}
+\DeclareSIUnit\molar{M}
+\DeclareSIUnit\gforce{\times{} g}
+
+%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
+% commands for lazy farts like me
 
 \newcommand{\mytitle}{
   \Large{
@@ -86,8 +110,14 @@
 
 \newcommand{\exvivo}{\textit{ex vivo}}
 
+\newcommand{\cd}[1]{CD{#1}}
+\newcommand{\anti}[1]{anti-{#1}}
+\newcommand{\anticd}[1]{\anti{\cd{#1}}}
+\newcommand{\cdp}[1]{\cd{#1}+}
+\newcommand{\cdn}[1]{\cd{#1}-}
+
 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
-% my environments
+% ditto for environments
 
 \newenvironment{mytitlepage}{
   \begin{singlespace}
@@ -99,7 +129,7 @@
 }
 
 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
-% document
+% begin document (proceed with caution)
 
 \begin{document}
 
@@ -208,15 +238,15 @@ Thank you to Lex Fridman and Devin Townsend for being awesome and inspirational.
 
 \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 anti-CD3 and CD28 \glspl{mab},
+approved T cell expansion technologies involve \anti-cd{3} and \anti-cd{28}
-usually mounted on magnetic beads. This method fails to recapitulate many key
+\glspl{mab}, usually mounted on magnetic beads. This method fails to
-signals found \invivo{} and is also heavily licensed by a few companies,
+recapitulate many key signals found \invivo{} and is also heavily licensed by a
-limiting its long-term usefulness to manufactures and clinicians. Furthermore,
+few companies, limiting its long-term usefulness to manufactures and clinicians.
-we understand that highly potent T cells are generally less-differentiated
+Furthermore, we understand that highly potent T cells are generally
-subtypes such as central memory and stem memory T cells. Despite this
+less-differentiated subtypes such as central memory and stem memory T cells.
-understanding, little has been done to optimize T cell expansion for generating
+Despite this understanding, little has been done to optimize T cell expansion
-these subtypes, including measurement and feedback control strategies that are
+for generating these subtypes, including measurement and feedback control
-necessary for any modern manufacturing process.
+strategies that are necessary for any modern manufacturing process.
 
 The goal of this thesis was to develop a microcarrier-based \gls{dms} T cell
 expansion system as well as determine biologically-meaningful \glspl{cqa} and
@@ -272,9 +302,9 @@ two genetically-modified \gls{car} T cell therapies against B cell malignancies.
 Despite these successes, \gls{car} T cell therapies are constrained by an
 expensive and difficult-to-scale manufacturing process with little control on
 cell quality and phenotype3,4. State-of-the-art T cell manufacturing techniques
-focus on anti-CD3 and anti-CD28 activation and expansion, typically presented on
+focus on \anticd{3} and \anticd{28} activation and expansion, typically
-superparamagnetic, iron-based microbeads (Invitrogen Dynabead, Miltenyi MACS
+presented on superparamagnetic, iron-based microbeads (Invitrogen Dynabead,
-beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers
+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
@@ -319,8 +349,8 @@ such as bioreactors.
 % TODO probably need to address some of the modeling stuff here as well
 
 This thesis describes a novel degradable microscaffold-based method derived from
-porous microcarriers functionalized with anti-CD3 and anti-CD28 \glspl{mab} for
+porous microcarriers functionalized with \anticd{3} and \anticd{28} \glspl{mab}
-use in T cell expansion cultures. Microcarriers have historically been used
+for use in T cell expansion cultures. Microcarriers have historically been used
 throughout the bioprocess industry for adherent cultures such as stem cells and
 \gls{cho} cells, but not with suspension cells such as T
 cells\cite{Heathman2015, Sart2011}. The microcarriers chosen to make the DMSs in
@@ -340,10 +370,10 @@ only provide superior expansion, but consistently provide a higher frequency of
 naïve/memory and CD4 T cells (CCR7+CD62L+) across multiple donors. We also
 demonstrate functional cytotoxicity using a CD19 \gls{car} and a superior
 performance, even at a lower \gls{car} T cell dose, of \gls{dms}-expanded
-\gls{car}-T cells \invivo{} in a mouse xenograft model of human B cell \gls{all}.
+\gls{car}-T cells \invivo{} in a mouse xenograft model of human B cell
-Our results indicate that \glspl{dms} provide a robust and scalable platform for
+\gls{all}. Our results indicate that \glspl{dms} provide a robust and scalable
-manufacturing therapeutic T cells with higher naïve/memory phenotype and more
+platform for manufacturing therapeutic T cells with higher naïve/memory
-balanced CD4+ T cell content.
+phenotype and more balanced CD4+ T cell content.
 
 \section*{hypothesis}
 
@@ -437,11 +467,11 @@ lymphoid organs where T cells normally expand. Typically, T cells are activated
 under close cell-cell contact via \glspl{apc} such as \glspl{dc}, which present
 peptide-\glspl{mhc} to T cells as well as a variety of other costimulatory
 signals. These close quarters allow for efficient autocrine/paracrine signaling
-among the expanding T cells, which secrete IL2 and other cytokines to assist
+among the expanding T cells, which secrete gls{il2} and other cytokines to
-their own growth. Additionally, the lymphoid tissues are comprised of \gls{ecm}
+assist their own growth. Additionally, the lymphoid tissues are comprised of
-components such as collagen, which provide signals to upregulate proliferation,
+\gls{ecm} components such as collagen, which provide signals to upregulate
-cytokine production, and pro-survival pathways\cite{Gendron2003, Ohtani2008,
+proliferation, cytokine production, and pro-survival pathways\cite{Gendron2003,
-  Boisvert2007, Ben-Horin2004}.
+  Ohtani2008, Boisvert2007, Ben-Horin2004}.
 
 A variety of solutions have been proposed to make the T cell expansion process
 more physiological. One strategy is to use modified feeder cell cultures to
@@ -590,18 +620,216 @@ technology for T cell manufacturing:
 \chapter{aim 1}\label{aim1}
 
 \section{introduction}
+
+The first aim was to develop a 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 anti-CD3 and anti-CD28 \glspl{mab} will
+provide superior expansion and memory phenotype compared to state-of-the-art
+bead-based T cell expansion technology.
+
+% TODO this doesn't flow that well and is repetitive with what comes above
+
+Microcarriers have been used throughout the bioprocess industry for adherent
+cell cultures such as \gls{cho} cells and stem cells, as they are able to
+achieve much greater surface area per unit volume than traditional 2D
+cultures\cite{Heathman2015, Sart2011}. Adding adhesive \glspl{mab} to the
+microcarriers will adapt them for suspension cell cultures such as T cells.
+Consequently, the large macroporous structure will allow T cells to cluster more
+closely, which in turn will enable better autocrine and paracrine signaling.
+Specifically, two cytokines that are secreted by T cells, IL-2 and IL-15, are
+known to drive expansion and memory phenotype respectively\cite{Buck2016}.
+Therefore, the proposed microcarrier system should enable greater expansion and
+better retention of memory phenotype compared to current bead-based methods.
+
 \section{methods}
+\subsection{dms functionalization}
+
+Gelatin microcarriers (\gls{cus} or \gls{cug}, GE Healthcare, DG-2001-OO and
+DG-0001-OO) were suspended at \SI{20}{\mg\per\ml} in 1X \gls{pbs} and
+autoclaved. All subsequent steps were done aseptically, and all reactions were
+carried out at \SI{20}{\mg\per\ml} carriers 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. \gls{snb} (Thermo Fisher 21217) was dissolved at approximately
+\SI{10}{\micro\molar} in sterile ultrapure water, and the true concentration was
+then determined using the \gls{haba} assay (see below).
+\SI{5}{\ul\of{\ab}\per\mL} \gls{pbs} 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 remaining in solution was quantified using 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.
+
+To coat with \gls{stp}, \SI{40}{\ug\per\mL} \gls{stp} (Jackson Immunoresearch
+016-000-114) was added and allowed to react for \SI{60}{\minute} at
+\SI{700}{RPM} of agitation. After the reaction, supernatant was taken for the
+\gls{bca} assay, and the carriers were washed analogously to the previous wash
+step to remove the biotin, except two washes were done and the agitation time
+was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3 (Biolegend
+317320) and CD28 (Biolegend 302904) were combined in a 1:1 mass ratio and added
+to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the
+\glspl{mab}, sterile \gls{bsa} (Sigma A9576) was added to a final concentration
+of \SI{2}{\percent} in order to prevent non-specific binding of the antibodies
+to the reaction tubes. \glspl{mab} were allowed to bind to the carriers for
+\SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, supernatants were
+sampled to quantify remaining antibody concentration using an \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}. They were washed once again in the cell culture media 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 {Table X}.
+
+%TODO this bit belongs in the next aim
+% In the case of the \gls{doe} experiment where
+% variable mAb surface density was utilized, the anti-CD3/anti-CD28 mAb mixture
+% was further combined with a biotinylated isotype control to reduce the overall
+% fraction of targeted mAbs (for example the 60\% mAb surface density corresponded
+% to 3 mass parts anti-CD3, 3 mass parts anti-CD8, and 4 mass parts isotype
+% control).
+
+\subsection{dms quality control assays}
+
+Biotin was quantified using the \gls{haba} assay (\gls{haba}/avidin premix from
+Sigma as product H2153-1VL). In the case of quantifying sulfo-NHS-biotin 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 \SI{70}{\ul}
+uCuvettes (BrandTech 759200). The extinction coefficient at \SI{500}{\nm} for
+\gls{haba}/avidin was assumed to be \SI{34000}{\per\cm\per\molar}.
+
+\gls{stp} binding to the carriers was quantified indirectly using a \gls{bca}
+kit (Thermo Fisher 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 Biotek plate reader.
+
+\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 antibodies used to coat the carriers were used as the standard for the
+\gls{elisa} standard curve.
+
+Open biotin binding sites on the \glspl{dms} after \gls{stp} coating was
+quantified indirectly using FITC-biotin (Thermo Fisher B10570). Briefly,
+\SI{400}{\pmol\per\ml} FITC-biotin were added to \gls{stp}-coated carriers and
+allowed to react for 20 min at room temperature under constant agitation. The
+supernatant was quantified against a standard curve of FITC-biotin using a
+Biotek plate reader.
+
+\Gls{stp} binding was verified after the \gls{stp}-binding step visually by
+adding biotin-FITC to the \gls{stp}-coated \glspl{dms}, resuspending in 1\%
+agarose gel, and imaging on a lightsheet microscope (Zeiss Z.1). \Gls{mab}
+binding was verified visually by first staining with \anti{gls{igg}}-FITC
+(Biolegend 406001), incubating for \SI{30}{\minute}, washing with \gls{pbs}, and
+imaging on a confocal microscope.
+
+\subsection{t cell culture}
+
+Cryopreserved primary human T cells were either obtained as sorted CD3
+subpopulations (Astarte Biotech) or isolated from \glspl{pbmc} (Zenbio) using a
+negative selection \gls{macs} kit for the CD3 subset (Miltenyi Biotech
+130-096-535). T cells were activated using \glspl{dms} or \SI{3.5}{\um} CD3/CD28
+magnetic beads (Miltenyi Biotech 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{2500}{\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. All media was serum-free Cell Therapy Systems OpTmizer (Thermo Fisher)
+or TexMACS (Miltentyi Biotech 170-076-307) supplemented with
+\SIrange{100}{400}{\IU\per\ml} \gls{rhil2} (Peprotech 200-02). 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 trypan blue or
+\gls{aopi} and a Countess Automated Cell Counter (Thermo Fisher). 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 ChemGlass glucometer.
+
+% this belongs in aim 2
+% In order to remove \glspl{dms} from
+% culture, collagenase D (Sigma Aldrich) was sterile filtered in culture media and
+% added to a final concentration of \SI{50}{\ug\per\ml} during media addition.
+
+Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul} \gls{stp}-PE
+(Biolegend 405204) and \SI{2}{ul} anti-CD45-AF647 (Biolegend 368538), incubating
+for an hour, and imaging on a spinning disk confocal microscope.
+
+\subsection{chemotaxis assay}
+
+Migratory function was assayed using a transwell chemotaxis assay as previously
+described62. Briefly, \SI{3e5}{\cell} were loaded into a transwell plate
+(\SI{5}{\um} pore size, Corning) with the basolateral chamber loaded with
+\SI{600}{\ul} media and 0, 250, or \SI{1000}{\ng\per\mL} 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
+countbright beads (Thermo Fisher C36950). The final readout was normalized using
+the \SI{0}{\ng\per\mL} concentration as background.
+
+\subsection{degranulation assay}
+
+Cytotoxicity of expanded CAR T cells was assessed using a degranulation assay as
+previously described63. Briefly, \num{3e5} T cells were incubated with
+\num{1.5e5} target cells consisting of either K562 wild type cells (ATCC) or
+CD19- expressing K562 cells transformed with \gls{crispr} (kindly provided by Dr.\
+Yvonne Chen, UCLA)64. Cells were seeded in a flat bottom 96 well plate with
+\SI{1}{\ug\per\ml} anti-CD49d (eBioscience 16-0499-81), \SI{2}{\micro\molar}
+monensin (eBioscience 00-4505-51), and \SI{1}{\ug\per\ml} anti-CD28 (eBioscience
+302914) (all \glspl{mab} functional grade) 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 CD8 fraction.
+
+\subsection{car expression}
+
+% TODO add acronym for PE
+\gls{car} expression was quantified as previously described65. Briefly, cells
+were washed once and stained with biotinylated Protein L (Thermo Fisher 29997).
+After a subsequent wash, cells were stained with PE-\gls{stp} (Biolegend
+405204), washed again, and analyzed on a BD Accuri. Readout was percent PE+
+cells as compared to secondary controls (PE-\gls{stp} with no Protein L).
+
+\subsection{car plasmid and lentiviral transduction}
+
+The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$ promotor29 was
+synthesized (Aldevron) and subcloned into a FUGW lentiviral transfer plasmid
+(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. To transduce primary human T cells, 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
+DMSs 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.
+
+% TODO add statistics section (anova, regression, and causal inference)
+
 \section{results}
 \section{discussion}
 
-\chapter{Aim 2}\label{aim2}
+\chapter{aim 2}\label{aim2}
 
 \section{introduction}
 \section{methods}
 \section{results}
 \section{discussion}
 
-\chapter{Aim 3}\label{aim3}
+\chapter{aim 3}\label{aim3}
 
 \section{introduction}
 \section{methods}