ndwarshuis
/
phd_thesis
1
0
Fork 0
Code Issues Pull Requests Packages Projects Releases Wiki Activity

ADD methods for aim 1

This commit is contained in:
Nathan Dwarshuis 2021-07-22 18:34:50 -04:00
parent bda13878e5
commit c785cd4bad
1 changed files with 265 additions and 37 deletions
Show Stats Download Patch File Download Diff File Expand all files Collapse all files

302
tex/thesis.tex
View File

@ -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}