From c785cd4badc5af9a5b29161bb0386233b291fcc1 Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Thu, 22 Jul 2021 18:34:50 -0400 Subject: [PATCH] ADD methods for aim 1 --- tex/thesis.tex | 302 +++++++++++++++++++++++++++++++++++++++++++------ 1 file changed, 265 insertions(+), 37 deletions(-) diff --git a/tex/thesis.tex b/tex/thesis.tex index 449a2ee..4d1b140 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -29,17 +29,11 @@ \setlist[description]{font=$\bullet$~\textbf\normalfont} -\sisetup{per-mode=symbol,list-units=single} -\DeclareSIUnit\activityunit{U} -\DeclareSIUnit\carrier{carriers} -\DeclareSIUnit\cell{cells} -\DeclareSIUnit\ab{mAbs} -\DeclareSIUnit\molar{M} -\DeclareSIUnit\gforce{\times{} g} +%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% +% acronyms for the lazy -% 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}, -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, -we understand that highly potent T cells are generally less-differentiated -subtypes such as central memory and stem memory T cells. 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. +approved T cell expansion technologies involve \anti-cd{3} and \anti-cd{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, we understand that highly potent T cells are generally +less-differentiated subtypes such as central memory and stem memory T cells. +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 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 -superparamagnetic, iron-based microbeads (Invitrogen Dynabead, Miltenyi MACS -beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers +focus on \anticd{3} and \anticd{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 @@ -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 -use in T cell expansion cultures. Microcarriers have historically been used +porous microcarriers functionalized with \anticd{3} and \anticd{28} \glspl{mab} +for use in T cell expansion cultures. Microcarriers have historically been used throughout the bioprocess industry for adherent cultures such as stem cells and \gls{cho} cells, but not with suspension cells such as T cells\cite{Heathman2015, Sart2011}. The microcarriers chosen to make the DMSs in @@ -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}. -Our results indicate that \glspl{dms} provide a robust and scalable platform for -manufacturing therapeutic T cells with higher naïve/memory phenotype and more -balanced CD4+ T cell content. +\gls{car}-T cells \invivo{} in a mouse xenograft model of human B cell +\gls{all}. Our results indicate that \glspl{dms} provide a robust and scalable +platform for manufacturing therapeutic T cells with higher naïve/memory +phenotype and more balanced CD4+ T cell content. \section*{hypothesis} @@ -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 -their own growth. Additionally, the lymphoid tissues are comprised of \gls{ecm} -components such as collagen, which provide signals to upregulate proliferation, -cytokine production, and pro-survival pathways\cite{Gendron2003, Ohtani2008, - Boisvert2007, Ben-Horin2004}. +among the expanding T cells, which secrete gls{il2} and other cytokines to +assist their own growth. Additionally, the lymphoid tissues are comprised of +\gls{ecm} components such as collagen, which provide signals to upregulate +proliferation, cytokine production, and pro-survival pathways\cite{Gendron2003, + Ohtani2008, Boisvert2007, Ben-Horin2004}. 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}