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tex/thesis.bbl
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@ -1,230 +0,0 @@
\begin{thebibliography}{10}
\expandafter\ifx\csname url\endcsname\relax
\def\url#1{\texttt{#1}}\fi
\expandafter\ifx\csname urlprefix\endcsname\relax\def\urlprefix{URL }\fi
\providecommand{\bibinfo}[2]{#2}
\providecommand{\eprint}[2][]{\url{#2}}
\bibitem{Fesnak2016}
\bibinfo{author}{Fesnak, A.~D.}, \bibinfo{author}{June, C.~H.} \&
\bibinfo{author}{Levine, B.~L.}
\newblock \bibinfo{title}{Engineered t cells: the promise and challenges of
cancer immunotherapy}.
\newblock \emph{\bibinfo{journal}{Nature Reviews Cancer}}
\textbf{\bibinfo{volume}{16}}, \bibinfo{pages}{566--581}
(\bibinfo{year}{2016}).
\bibitem{Rosenberg2015}
\bibinfo{author}{Rosenberg, S.~A.} \& \bibinfo{author}{Restifo, N.~P.}
\newblock \bibinfo{title}{Adoptive cell transfer as personalized immunotherapy
for human cancer}.
\newblock \emph{\bibinfo{journal}{Science}} \textbf{\bibinfo{volume}{348}},
\bibinfo{pages}{62--68} (\bibinfo{year}{2015}).
\bibitem{Roddie2019}
\bibinfo{author}{Roddie, C.}, \bibinfo{author}{O'Reilly, M.},
\bibinfo{author}{Pinto, J. D.~A.}, \bibinfo{author}{Vispute, K.} \&
\bibinfo{author}{Lowdell, M.}
\newblock \bibinfo{title}{Manufacturing chimeric antigen receptor t cells:
issues and challenges}.
\newblock \emph{\bibinfo{journal}{Cytotherapy}} (\bibinfo{year}{2019}).
\bibitem{Dwarshuis2017}
\bibinfo{author}{Dwarshuis, N.~J.}, \bibinfo{author}{Parratt, K.},
\bibinfo{author}{Santiago-Miranda, A.} \& \bibinfo{author}{Roy, K.}
\newblock \bibinfo{title}{Cells as advanced therapeutics: State-of-the-art,
challenges, and opportunities in large scale biomanufacturing of high-quality
cells for adoptive immunotherapies}.
\newblock \emph{\bibinfo{journal}{Advanced Drug Delivery Reviews}}
\textbf{\bibinfo{volume}{114}}, \bibinfo{pages}{222--239}
(\bibinfo{year}{2017}).
\bibitem{Wang2016}
\bibinfo{author}{Wang, X.} \& \bibinfo{author}{Rivi{\`{e}}re, I.}
\newblock \bibinfo{title}{Clinical manufacturing of {CAR} t cells: foundation
of a promising therapy}.
\newblock \emph{\bibinfo{journal}{Molecular Therapy - Oncolytics}}
\textbf{\bibinfo{volume}{3}}, \bibinfo{pages}{16015} (\bibinfo{year}{2016}).
\bibitem{Piscopo2017}
\bibinfo{author}{Piscopo, N.~J.} \emph{et~al.}
\newblock \bibinfo{title}{Bioengineering solutions for manufacturing challenges
in {CAR} t cells}.
\newblock \emph{\bibinfo{journal}{Biotechnology Journal}}
\textbf{\bibinfo{volume}{13}}, \bibinfo{pages}{1700095}
(\bibinfo{year}{2017}).
\bibitem{Bashour2015}
\bibinfo{author}{Bashour, K.~T.} \emph{et~al.}
\newblock \bibinfo{title}{Functional characterization of a t cell stimulation
reagent for the production of therapeutic chimeric antigen receptor t cells}.
\newblock \emph{\bibinfo{journal}{Blood}} \textbf{\bibinfo{volume}{126}},
\bibinfo{pages}{1901--1901} (\bibinfo{year}{2015}).
\bibitem{Gendron2003}
\bibinfo{author}{Gendron, S.}, \bibinfo{author}{Couture, J.} \&
\bibinfo{author}{Aoudjit, F.}
\newblock \bibinfo{title}{Integrin $\alpha$2$\beta$1inhibits fas-mediated
apoptosis in t lymphocytes by protein phosphatase 2a-dependent activation of
the {MAPK}/{ERK} pathway}.
\newblock \emph{\bibinfo{journal}{Journal of Biological Chemistry}}
\textbf{\bibinfo{volume}{278}}, \bibinfo{pages}{48633--48643}
(\bibinfo{year}{2003}).
\bibitem{Ohtani2008}
\bibinfo{author}{Ohtani, O.} \& \bibinfo{author}{Ohtani, Y.}
\newblock \bibinfo{title}{Structure and function of rat lymph nodes}.
\newblock \emph{\bibinfo{journal}{Archives of Histology and Cytology}}
\textbf{\bibinfo{volume}{71}}, \bibinfo{pages}{69--76}
(\bibinfo{year}{2008}).
\bibitem{Boisvert2007}
\bibinfo{author}{Boisvert, M.}, \bibinfo{author}{Gendron, S.},
\bibinfo{author}{Chetoui, N.} \& \bibinfo{author}{Aoudjit, F.}
\newblock \bibinfo{title}{Alpha2beta1 integrin signaling augments t cell
receptor-dependent production of interferon-gamma in human t cells}.
\newblock \emph{\bibinfo{journal}{Molecular Immunology}}
\textbf{\bibinfo{volume}{44}}, \bibinfo{pages}{3732--3740}
(\bibinfo{year}{2007}).
\bibitem{Ben-Horin2004}
\bibinfo{author}{Ben-Horin, S.} \& \bibinfo{author}{Bank, I.}
\newblock \bibinfo{title}{The role of very late antigen-1 in immune-mediated
inflammation}.
\newblock \emph{\bibinfo{journal}{Clinical Immunology}}
\textbf{\bibinfo{volume}{113}}, \bibinfo{pages}{119--129}
(\bibinfo{year}{2004}).
\bibitem{Forget2014}
\bibinfo{author}{Forget, M.-A.} \emph{et~al.}
\newblock \bibinfo{title}{Activation and propagation of tumor-infiltrating
lymphocytes on clinical-grade designer artificial antigen-presenting cells
for adoptive immunotherapy of melanoma}.
\newblock \emph{\bibinfo{journal}{Journal of Immunotherapy}}
\textbf{\bibinfo{volume}{37}}, \bibinfo{pages}{448--460}
(\bibinfo{year}{2014}).
\bibitem{Cheung2018}
\bibinfo{author}{Cheung, A.~S.}, \bibinfo{author}{Zhang, D. K.~Y.},
\bibinfo{author}{Koshy, S.~T.} \& \bibinfo{author}{Mooney, D.~J.}
\newblock \bibinfo{title}{Scaffolds that mimic antigen-presenting cells enable
ex vivo expansion of primary {T} cells}.
\newblock \emph{\bibinfo{journal}{Nature Biotechnology}}
\textbf{\bibinfo{volume}{36}}, \bibinfo{pages}{160--169}
(\bibinfo{year}{2018}).
\bibitem{Rio2018}
\bibinfo{author}{del R{\'{\i}}o, E.~P.}, \bibinfo{author}{Miguel, M.~M.},
\bibinfo{author}{Veciana, J.}, \bibinfo{author}{Ratera, I.} \&
\bibinfo{author}{Guasch, J.}
\newblock \bibinfo{title}{Artificial 3d culture systems for t cell expansion}.
\newblock \emph{\bibinfo{journal}{{ACS} Omega}} \textbf{\bibinfo{volume}{3}},
\bibinfo{pages}{5273--5280} (\bibinfo{year}{2018}).
\bibitem{Delalat2017}
\bibinfo{author}{Delalat, B.} \emph{et~al.}
\newblock \bibinfo{title}{{3D printed lattices as an activation and expansion
platform for T cell therapy}}.
\newblock \emph{\bibinfo{journal}{Biomaterials}}
\textbf{\bibinfo{volume}{140}}, \bibinfo{pages}{58--68}
(\bibinfo{year}{2017}).
\bibitem{meyer15_immun}
\bibinfo{author}{Meyer, R.~A.} \emph{et~al.}
\newblock \bibinfo{title}{Immunoengineering: Biodegradable nanoellipsoidal
artificial antigen presenting cells for antigen specific t-cell activation
(small 13/2015)}.
\newblock \emph{\bibinfo{journal}{Small}} \textbf{\bibinfo{volume}{11}},
\bibinfo{pages}{1612--1612} (\bibinfo{year}{2015}).
\bibitem{Lambert2017}
\bibinfo{author}{Lambert, L.~H.} \emph{et~al.}
\newblock \bibinfo{title}{{Improving T Cell Expansion with a Soft Touch.}}
\newblock \emph{\bibinfo{journal}{Nano letters}} \textbf{\bibinfo{volume}{17}},
\bibinfo{pages}{821--826} (\bibinfo{year}{2017}).
\bibitem{Xu2014}
\bibinfo{author}{Xu, Y.} \emph{et~al.}
\newblock \bibinfo{title}{Closely related t-memory stem cells correlate with in
vivo expansion of car.cd19-t cells and are preserved by il-7 and il-15.}
\newblock \emph{\bibinfo{journal}{Blood}} \textbf{\bibinfo{volume}{123}},
\bibinfo{pages}{3750--3759} (\bibinfo{year}{2014}).
\bibitem{Fraietta2018}
\bibinfo{author}{Fraietta, J.~A.} \emph{et~al.}
\newblock \bibinfo{title}{Determinants of response and resistance to {CD}19
chimeric antigen receptor ({CAR}) t cell therapy of chronic lymphocytic
leukemia}.
\newblock \emph{\bibinfo{journal}{Nature Medicine}}
\textbf{\bibinfo{volume}{24}}, \bibinfo{pages}{563--571}
(\bibinfo{year}{2018}).
\bibitem{Gattinoni2011}
\bibinfo{author}{Gattinoni, L.} \emph{et~al.}
\newblock \bibinfo{title}{A human memory t cell subset with stem
cell{\textendash}like properties}.
\newblock \emph{\bibinfo{journal}{Nature Medicine}}
\textbf{\bibinfo{volume}{17}}, \bibinfo{pages}{1290--1297}
(\bibinfo{year}{2011}).
\bibitem{Gattinoni2012}
\bibinfo{author}{Gattinoni, L.}, \bibinfo{author}{Klebanoff, C.~A.} \&
\bibinfo{author}{Restifo, N.~P.}
\newblock \bibinfo{title}{{Paths to stemness: building the ultimate antitumour
T cell.}}
\newblock \emph{\bibinfo{journal}{Nature reviews. Cancer}}
\textbf{\bibinfo{volume}{12}}, \bibinfo{pages}{671--84}
(\bibinfo{year}{2012}).
\bibitem{Wang2018}
\bibinfo{author}{Wang, D.} \emph{et~al.}
\newblock \bibinfo{title}{Glioblastoma-targeted {CD}4+ {CAR} t cells mediate
superior antitumor activity}.
\newblock \emph{\bibinfo{journal}{{JCI} Insight}} \textbf{\bibinfo{volume}{3}}
(\bibinfo{year}{2018}).
\bibitem{Yang2017}
\bibinfo{author}{Yang, Y.} \emph{et~al.}
\newblock \bibinfo{title}{{TCR} engagement negatively affects {CD}8 but not
{CD}4 {CAR} t cell expansion and leukemic clearance}.
\newblock \emph{\bibinfo{journal}{Science Translational Medicine}}
\textbf{\bibinfo{volume}{9}}, \bibinfo{pages}{eaag1209}
(\bibinfo{year}{2017}).
\bibitem{Heathman2015}
\bibinfo{author}{Heathman, T. R.~J.} \emph{et~al.}
\newblock \bibinfo{title}{Expansion, harvest and cryopreservation of human
mesenchymal stem cells in a serum-free microcarrier process}.
\newblock \emph{\bibinfo{journal}{Biotechnology and Bioengineering}}
\textbf{\bibinfo{volume}{112}}, \bibinfo{pages}{1696--1707}
(\bibinfo{year}{2015}).
\bibitem{Sart2011}
\bibinfo{author}{Sart, S.}, \bibinfo{author}{Errachid, A.},
\bibinfo{author}{Schneider, Y.-J.} \& \bibinfo{author}{Agathos, S.~N.}
\newblock \bibinfo{title}{Controlled expansion and differentiation of
mesenchymal stem cells in a microcarrier based stirred bioreactor}.
\newblock \emph{\bibinfo{journal}{{BMC} Proceedings}}
\textbf{\bibinfo{volume}{5}} (\bibinfo{year}{2011}).
\bibitem{Buck2016}
\bibinfo{author}{Buck, M.~D.} \emph{et~al.}
\newblock \bibinfo{title}{{Mitochondrial Dynamics Controls T Cell Fate through
Metabolic Programming}}.
\newblock \emph{\bibinfo{journal}{Cell}} \textbf{\bibinfo{volume}{166}},
\bibinfo{pages}{114} (\bibinfo{year}{2016}).
\bibitem{van_der_Windt_2012}
\bibinfo{author}{van~der Windt, G.~J.} \emph{et~al.}
\newblock \bibinfo{title}{Mitochondrial respiratory capacity is a critical
regulator of {CD}8+ t cell memory development}.
\newblock \emph{\bibinfo{journal}{Immunity}} \textbf{\bibinfo{volume}{36}},
\bibinfo{pages}{68--78} (\bibinfo{year}{2012}).
\bibitem{Spitzer2016}
\bibinfo{author}{Spitzer, M.~H.} \& \bibinfo{author}{Nolan, G.~P.}
\newblock \bibinfo{title}{Mass cytometry: Single cells, many features}.
\newblock \emph{\bibinfo{journal}{Cell}} \textbf{\bibinfo{volume}{165}},
\bibinfo{pages}{780--791} (\bibinfo{year}{2016}).
\end{thebibliography}

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tex/thesis.tex
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@ -65,6 +65,10 @@
\newacronym{macs}{MACS}{magnetic activated cell sorting} \newacronym{macs}{MACS}{magnetic activated cell sorting}
\newacronym{aopi}{AO/PI}{acridine orange/propidium iodide} \newacronym{aopi}{AO/PI}{acridine orange/propidium iodide}
\newacronym{igg}{IgG}{immunoglobulin G} \newacronym{igg}{IgG}{immunoglobulin G}
\newacronym{pe}{PE}{phycoerythrin}
\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 \newacronym{crispr}{CRISPR}{clustered regularly interspaced short
palindromic repeats} palindromic repeats}
@ -105,17 +109,24 @@
} }
\newcommand{\invivo}{\textit{in vivo}} \newcommand{\invivo}{\textit{in vivo}}
\newcommand{\invitro}{\textit{in vitro}} \newcommand{\invitro}{\textit{in vitro}}
\newcommand{\exvivo}{\textit{ex vivo}} \newcommand{\exvivo}{\textit{ex vivo}}
\newcommand{\cd}[1]{CD{#1}} \newcommand{\cd}[1]{CD{#1}}
\newcommand{\anti}[1]{anti-{#1}} \newcommand{\anti}[1]{anti-{#1}}
\newcommand{\anticd}[1]{\anti{\cd{#1}}} \newcommand{\acd}[1]{\anti{\cd{#1}}}
\newcommand{\cdp}[1]{\cd{#1}+} \newcommand{\cdp}[1]{\cd{#1}+}
\newcommand{\cdn}[1]{\cd{#1}-} \newcommand{\cdn}[1]{\cd{#1}-}
\newcommand{\catnum}[2]{(#1, #2)}
\newcommand{\product}[3]{#1 \catnum{#2}{#3}}
\newcommand{\thermo}{Thermo Fisher}
\newcommand{\miltenyi}{Miltenyi Biotech}
\newcommand{\bl}{Biolegend}
\newcommand{\inlinecode}{\texttt}
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% ditto for environments % ditto for environments
@ -302,7 +313,7 @@ two genetically-modified \gls{car} T cell therapies against B cell malignancies.
Despite these successes, \gls{car} T cell therapies are constrained by an Despite these successes, \gls{car} T cell therapies are constrained by an
expensive and difficult-to-scale manufacturing process with little control on expensive and difficult-to-scale manufacturing process with little control on
cell quality and phenotype3,4. State-of-the-art T cell manufacturing techniques cell quality and phenotype3,4. State-of-the-art T cell manufacturing techniques
focus on \anticd{3} and \anticd{28} activation and expansion, typically focus on \acd{3} and \acd{28} activation and expansion, typically
presented on superparamagnetic, iron-based microbeads (Invitrogen Dynabead, presented on superparamagnetic, iron-based microbeads (Invitrogen Dynabead,
Miltenyi MACS 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}. (Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017, Bashour2015}.
@ -349,7 +360,7 @@ such as bioreactors.
% TODO probably need to address some of the modeling stuff here as well % TODO probably need to address some of the modeling stuff here as well
This thesis describes a novel degradable microscaffold-based method derived from This thesis describes a novel degradable microscaffold-based method derived from
porous microcarriers functionalized with \anticd{3} and \anticd{28} \glspl{mab} porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab}
for 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 throughout the bioprocess industry for adherent cultures such as stem cells and
\gls{cho} cells, but not with suspension cells such as T \gls{cho} cells, but not with suspension cells such as T
@ -458,7 +469,7 @@ successes, \gls{car} T cell therapies are constrained by an expensive and
difficult-to-scale manufacturing process\cite{Roddie2019, Dwarshuis2017}. difficult-to-scale manufacturing process\cite{Roddie2019, Dwarshuis2017}.
Of critical concern, state-of-the-art manufacturing techniques focus only on Of critical concern, state-of-the-art manufacturing techniques focus only on
Signal 1 and Signal 2-based activation via anti-CD3 and anti-CD28 \glspl{mab}, Signal 1 and Signal 2-based activation via \acd{3} and \acd{28} \glspl{mab},
typically presented on a microbead (Invitrogen Dynabead, Miltenyi MACS beads) or typically presented on a microbead (Invitrogen Dynabead, Miltenyi MACS beads) or
nanobead (Miltenyi TransACT), but also in soluble forms in the case of antibody nanobead (Miltenyi TransACT), but also in soluble forms in the case of antibody
tetramers (Expamer)\cite{Wang2016, Piscopo2017, Roddie2019, Bashour2015}. These tetramers (Expamer)\cite{Wang2016, Piscopo2017, Roddie2019, Bashour2015}. These
@ -497,8 +508,8 @@ cytokine release properties and ability to resist exhaustion\cite{Wang2018,
Yang2017}, and no method exists to preferentially expand the CD4 population Yang2017}, and no method exists to preferentially expand the CD4 population
compared to state-of-the-art systems. compared to state-of-the-art systems.
Here we propose a method using microcarriers functionalized with anti-CD3 and Here we propose a method using microcarriers functionalized with \acd{3} and
anti-CD28 \glspl{mab} for use in T cell expansion cultures. Microcarriers have \acd{28} \glspl{mab} for use in T cell expansion cultures. Microcarriers have
historically been used throughout the bioprocess industry for adherent cultures 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 such as stem cells and \gls{cho} cells, but not with suspension cells such as T
cells\cite{Heathman2015, Sart2011}. The carriers have a macroporous structure cells\cite{Heathman2015, Sart2011}. The carriers have a macroporous structure
@ -625,7 +636,7 @@ The first aim was to develop a microcarrier system that mimics several key
aspects of the \invivo{} lymph node microenvironment. We compared compare this aspects of the \invivo{} lymph node microenvironment. We compared compare this
system to state-of-the-art T cell activation technologies for both expansion system to state-of-the-art T cell activation technologies for both expansion
potential and memory cell formation. The governing hypothesis was that potential and memory cell formation. The governing hypothesis was that
microcarriers functionalized with anti-CD3 and anti-CD28 \glspl{mab} will microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} will
provide superior expansion and memory phenotype compared to state-of-the-art provide superior expansion and memory phenotype compared to state-of-the-art
bead-based T cell expansion technology. bead-based T cell expansion technology.
@ -652,9 +663,9 @@ 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 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, 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 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 volume ratio. \product{\Gls{snb}}{\thermo}{21217} was dissolved at
\SI{10}{\micro\molar} in sterile ultrapure water, and the true concentration was approximately \SI{10}{\micro\molar} in sterile ultrapure water, and the true
then determined using the \gls{haba} assay (see below). 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 \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 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 reaction, the amount of biotin remaining in solution was quantified using the
@ -665,23 +676,23 @@ entailed adding sterile \gls{pbs} in a 10:1 volumetric ratio, agitating at
\SI{1000}{\gforce} for \SI{1}{\minute}, and removing all liquid back down to the \SI{1000}{\gforce} for \SI{1}{\minute}, and removing all liquid back down to the
reaction volume. reaction volume.
To coat with \gls{stp}, \SI{40}{\ug\per\mL} \gls{stp} (Jackson Immunoresearch To coat with \gls{stp}, \SI{40}{\ug\per\mL} \product{\gls{stp}}{Jackson
016-000-114) was added and allowed to react for \SI{60}{\minute} at Immunoresearch}{ 016-000-114} was added and allowed to react for
\SI{700}{RPM} of agitation. After the reaction, supernatant was taken for the \SI{60}{\minute} at \SI{700}{RPM} of agitation. After the reaction, supernatant
\gls{bca} assay, and the carriers were washed analogously to the previous wash was taken for the \gls{bca} assay, and the carriers were washed analogously to
step to remove the biotin, except two washes were done and the agitation time the previous wash step to remove the biotin, except two washes were done and the
was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3 (Biolegend agitation time was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3
317320) and CD28 (Biolegend 302904) were combined in a 1:1 mass ratio and added \catnum{\bl}{317320} and CD28 \catnum{\bl}{302904} were combined in a 1:1 mass
to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the ratio and added to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along
\glspl{mab}, sterile \gls{bsa} (Sigma A9576) was added to a final concentration with the \glspl{mab}, sterile \product{\gls{bsa}}{Sigma}{A9576} was added to a
of \SI{2}{\percent} in order to prevent non-specific binding of the antibodies final concentration of \SI{2}{\percent} in order to prevent non-specific binding
to the reaction tubes. \glspl{mab} were allowed to bind to the carriers for of the antibodies to the reaction tubes. \glspl{mab} were allowed to bind to the
\SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, supernatants were carriers for \SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding,
sampled to quantify remaining antibody concentration using an \anti{\gls{igg}} supernatants were sampled to quantify remaining \gls{mab} concentration using an
\gls{elisa} kit (Abcam 157719). Fully functionalized \glspl{dms} were washed in \product{\anti{\gls{igg}} \gls{elisa} kit}{Abcam}{157719}. Fully functionalized
sterile \gls{pbs} analogous to the previous washing step to remove excess \glspl{dms} were washed in sterile \gls{pbs} analogous to the previous washing
\gls{stp}. They were washed once again in the cell culture media to be used for step to remove excess \gls{stp}. They were washed once again in the cell culture
the T cell expansion. media to be used for the T cell expansion.
The concentration of the final \gls{dms} suspension was found by taking a 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 \SI{50}{\uL} sample, plating in a well, and imaging the entire well. The image
@ -699,21 +710,21 @@ was then manually counted to obtain a concentration. Surface area for
\subsection{dms quality control assays} \subsection{dms quality control assays}
Biotin was quantified using the \gls{haba} assay (\gls{haba}/avidin premix from Biotin was quantified using the \product{\gls{haba} assay}{Sigma}{H2153-1VL}. In
Sigma as product H2153-1VL). In the case of quantifying sulfo-NHS-biotin prior the case of quantifying \gls{snb} prior to adding it to the microcarriers, the
to adding it to the microcarriers, the sample volume was quenched in a 1:1 sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar} NaOH
volumetric ratio with \SI{1}{\molar} NaOH and allowed to react for and allowed to react for \SI{1}{\minute} in order to prevent the reactive ester
\SI{1}{\minute} in order to prevent the reactive ester linkages from binding to linkages from binding to the avidin proteins in the \gls{haba}/avidin premix.
the avidin proteins in the \gls{haba}/avidin premix. All quantifications of All quantifications of \gls{haba} were performed on an Eppendorf D30
\gls{haba} were performed on an Eppendorf D30 Spectrophotometer using \SI{70}{\ul} Spectrophotometer using \product{\SI{70}{\ul} cuvettes}{BrandTech}{759200}. The
uCuvettes (BrandTech 759200). The extinction coefficient at \SI{500}{\nm} for extinction coefficient at \SI{500}{\nm} for \gls{haba}/avidin was assumed to be
\gls{haba}/avidin was assumed to be \SI{34000}{\per\cm\per\molar}. \SI{34000}{\per\cm\per\molar}.
\gls{stp} binding to the carriers was quantified indirectly using a \gls{bca} \gls{stp} binding to the carriers was quantified indirectly using a
kit (Thermo Fisher 23227) according to the manufacturer’s instructions, with the \product{\gls{bca} kit }{\thermo}{23227} according to the manufacturer’s
exception that the standard curve was made with known concentrations of purified instructions, with the exception that the standard curve was made with known
\gls{stp} instead of \gls{bsa}. Absorbance at \SI{592}{\nm} was concentrations of purified \gls{stp} instead of \gls{bsa}. Absorbance at
quantified using a Biotek plate reader. \SI{592}{\nm} was quantified using a Biotek plate reader.
\Gls{mab} binding to the microcarriers was quantified indirectly using an \Gls{mab} binding to the microcarriers was quantified indirectly using an
\gls{elisa} assay per the manufacturer’s instructions, with the exception that \gls{elisa} assay per the manufacturer’s instructions, with the exception that
@ -721,103 +732,132 @@ the same antibodies used to coat the carriers were used as the standard for the
\gls{elisa} standard curve. \gls{elisa} standard curve.
Open biotin binding sites on the \glspl{dms} after \gls{stp} coating was Open biotin binding sites on the \glspl{dms} after \gls{stp} coating was
quantified indirectly using FITC-biotin (Thermo Fisher B10570). Briefly, quantified indirectly using \product{FITC-biotin}{\thermo}{B10570}.
\SI{400}{\pmol\per\ml} FITC-biotin were added to \gls{stp}-coated carriers and Briefly, \SI{400}{\pmol\per\ml} FITC-biotin were added to \gls{stp}-coated
allowed to react for 20 min at room temperature under constant agitation. The carriers and allowed to react for \SI{20}{\minute} at room temperature under
supernatant was quantified against a standard curve of FITC-biotin using a constant agitation. The supernatant was quantified against a standard curve of
Biotek plate reader. FITC-biotin using a Biotek plate reader.
\Gls{stp} binding was verified after the \gls{stp}-binding step visually by \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\% adding biotin-FITC to the \gls{stp}-coated \glspl{dms}, resuspending in
agarose gel, and imaging on a lightsheet microscope (Zeiss Z.1). \Gls{mab} \SI{1}{\percent} agarose gel, and imaging on a \product{lightsheet
binding was verified visually by first staining with \anti{gls{igg}}-FITC microscope}{Zeiss}{Z.1}. \Gls{mab} binding was verified visually by first
(Biolegend 406001), incubating for \SI{30}{\minute}, washing with \gls{pbs}, and staining with \product{\anti{gls{igg}}-FITC}{\bl}{406001}, incubating for
imaging on a confocal microscope. \SI{30}{\minute}, washing with \gls{pbs}, and imaging on a confocal microscope.
\subsection{t cell culture} \subsection{t cell culture}
Cryopreserved primary human T cells were either obtained as sorted CD3 % TODO verify countess product number
subpopulations (Astarte Biotech) or isolated from \glspl{pbmc} (Zenbio) using a Cryopreserved primary human T cells were either obtained as sorted
negative selection \gls{macs} kit for the CD3 subset (Miltenyi Biotech \product{\cdp{3} T cells}{Astarte Biotech}{1017} or isolated from
130-096-535). T cells were activated using \glspl{dms} or \SI{3.5}{\um} CD3/CD28 \product{\glspl{pbmc}}{Zenbio}{SER-PBMC} using a negative selection
magnetic beads (Miltenyi Biotech 130-091-441). In the case of beads, T cells \product{\cdp{3} \gls{macs} kit}{\miltenyi}{130-096-535}. T cells were activated
were activated at the manufacturer recommended cell:bead ratio of 2:1. In the using \glspl{dms} or \product{\SI{3.5}{\um} CD3/CD28 magnetic
case of \glspl{dms}, cells were activated using \SI{2500}{\dms\per\cm\squared} beads}{\miltenyi}{130-091-441}. In the case of beads, T cells were activated
unless otherwise noted. Initial cell density was at the manufacturer recommended cell:bead ratio of 2:1. In the case of
\SIrange{2e6}{2.5e6}{\cell\per\ml} to in a 96 well plate with \SI{300}{\ul} \glspl{dms}, cells were activated using \SI{2500}{\dms\per\cm\squared} unless
volume. All media was serum-free Cell Therapy Systems OpTmizer (Thermo Fisher) otherwise noted. Initial cell density was \SIrange{2e6}{2.5e6}{\cell\per\ml} to
or TexMACS (Miltentyi Biotech 170-076-307) supplemented with in a 96 well plate with \SI{300}{\ul} volume. Serum-free media was either
\SIrange{100}{400}{\IU\per\ml} \gls{rhil2} (Peprotech 200-02). Cell cultures \product{OpTmizer}{\thermo}{A1048501} or
were expanded for \SI{14}{\day} as counted from the time of initial seeding and \product{TexMACS}{\miltenyi}{170-076-307} supplemented with
activation. Cell counts and viability were assessed using trypan blue or \SIrange{100}{400}{\IU\per\ml} \product{\gls{rhil2}}{Peprotech}{200-02}. Cell
\gls{aopi} and a Countess Automated Cell Counter (Thermo Fisher). Media was cultures were expanded for \SI{14}{\day} as counted from the time of initial
added to cultures every \SIrange{2}{3}{\day} depending on media color or a seeding and activation. Cell counts and viability were assessed using
\SI{300}{\mg\per\deci\liter} minimum glucose threshold. Media glucose was \product{trypan blue}{\thermo}{T10282} or \product{\gls{aopi}}{Nexcelom
measured using a ChemGlass glucometer. 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}.
% this belongs in aim 2 % TODO this belongs in aim 2
% In order to remove \glspl{dms} from % In order to remove \glspl{dms} from
% culture, collagenase D (Sigma Aldrich) was sterile filtered in culture media and % 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. % 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 Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul}
(Biolegend 405204) and \SI{2}{ul} anti-CD45-AF647 (Biolegend 368538), incubating \product{\gls{stp}-\gls{pe}}{\bl}{405204} and \SI{2}{ul}
for an hour, and imaging on a spinning disk confocal microscope. \product{\acd{45}-\gls{af647}}{\bl}{368538}, incubating for \SI{1}{\hour}, and
imaging on a spinning disk confocal microscope.
\subsection{chemotaxis assay} \subsection{chemotaxis assay}
% TODO not sure about the transwell product number
Migratory function was assayed using a transwell chemotaxis assay as previously Migratory function was assayed using a transwell chemotaxis assay as previously
described62. Briefly, \SI{3e5}{\cell} were loaded into a transwell plate described\cite{Hromas1997}. Briefly, \SI{3e5}{\cell} were loaded into a
(\SI{5}{\um} pore size, Corning) with the basolateral chamber loaded with \product{transwell plate with \SI{5}{\um} pore size}{Corning}{3421} with the
\SI{600}{\ul} media and 0, 250, or \SI{1000}{\ng\per\mL} CCL21 (Peprotech basolateral chamber loaded with \SI{600}{\ul} media and 0, 250, or
250-13). The plate was incubated for \SI{4}{\hour} after loading, and the \SI{1000}{\ng\per\mL} \product{CCL21}{Peprotech}{250-13}. The plate was
basolateral chamber of each transwell was quantified for total cells using incubated for \SI{4}{\hour} after loading, and the basolateral chamber of each
countbright beads (Thermo Fisher C36950). The final readout was normalized using transwell was quantified for total cells using \product{countbright
the \SI{0}{\ng\per\mL} concentration as background. beads}{\thermo}{C36950}. The final readout was normalized using the
\SI{0}{\ng\per\mL} concentration as background.
\subsection{degranulation assay} \subsection{degranulation assay}
Cytotoxicity of expanded CAR T cells was assessed using a degranulation assay as Cytotoxicity of expanded \gls{car} T cells was assessed using a degranulation
previously described63. Briefly, \num{3e5} T cells were incubated with assay as previously described\cite{Schmoldt1975}. Briefly, \num{3e5} T cells
\num{1.5e5} target cells consisting of either K562 wild type cells (ATCC) or were incubated with \num{1.5e5} target cells consisting of either \product{K562
CD19- expressing K562 cells transformed with \gls{crispr} (kindly provided by Dr.\ wild type cells}{ATCC}{CCL-243} or CD19- expressing K562 cells transformed
Yvonne Chen, UCLA)64. Cells were seeded in a flat bottom 96 well plate with with \gls{crispr} (kindly provided by Dr.\ Yvonne Chen, UCLA)\cite{Zah2016}.
\SI{1}{\ug\per\ml} anti-CD49d (eBioscience 16-0499-81), \SI{2}{\micro\molar} Cells were seeded in a flat bottom 96 well plate with \SI{1}{\ug\per\ml}
monensin (eBioscience 00-4505-51), and \SI{1}{\ug\per\ml} anti-CD28 (eBioscience \product{\acd{49d}}{eBioscience}{16-0499-81}, \SI{2}{\micro\molar} \product{monensin}{eBioscience}{
302914) (all \glspl{mab} functional grade) with \SI{250}{\ul} total volume. 00-4505-51}, and \SI{1}{\ug\per\ml} \product{\acd{28}}{eBioscience}{302914} (all
After \SI{4}{\hour} incubation at \SI{37}{\degreeCelsius}, cells were stained functional grade \glspl{mab}) with \SI{250}{\ul} total volume. After
for CD3, CD4, and CD107a and analyzed on a BD LSR Fortessa. Readout was \SI{4}{\hour} incubation at \SI{37}{\degreeCelsius}, cells were stained for CD3,
calculated as the percent \cdp{107a} cells of the total CD8 fraction. 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} \subsection{car expression}
% TODO add acronym for PE \gls{car} expression was quantified as previously described\cite{Zheng2012}.
\gls{car} expression was quantified as previously described65. Briefly, cells Briefly, cells were washed once and stained with \product{biotinylated
were washed once and stained with biotinylated Protein L (Thermo Fisher 29997). \gls{ptnl}}{\thermo}{29997}. After a subsequent wash, cells were stained with
After a subsequent wash, cells were stained with PE-\gls{stp} (Biolegend \product{\gls{pe}-\gls{stp}}{\bl}{405204}, washed again, and analyzed on a
405204), washed again, and analyzed on a BD Accuri. Readout was percent PE+ BD Accuri. Readout was percent \gls{pe}+ cells as compared to secondary controls
cells as compared to secondary controls (PE-\gls{stp} with no Protein L). (\gls{pe}-\gls{stp} with no \gls{ptnl}).
\subsection{car plasmid and lentiviral transduction} \subsection{car plasmid and lentiviral transduction}
The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$ promotor29 was The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$
synthesized (Aldevron) and subcloned into a FUGW lentiviral transfer plasmid promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a
(Emory Viral Vector Core). Lentiviral vectors were synthesized by the Emory \product{FUGW}{Addgene}{14883} kindly provided by the Emory Viral Vector Core.
Viral Vector Core or the Cincinnati Children's Hospital Medical Center Viral Lentiviral vectors were synthesized by the Emory Viral Vector Core or the
Vector Core. To transduce primary human T cells, retronectin (Takara T100A) was Cincinnati Children's Hospital Medical Center Viral Vector Core. To transduce
coated onto non-TC treated 96 well plates and used to immobilize lentiviral primary human T cells, \product{retronectin}{Takara}{T100A} was coated onto
vector particles according to the manufacturer's instructions. Briefly, non-TC treated 96 well plates and used to immobilize lentiviral vector particles
retronectin solution was adsorbed overnight at \SI{4}{\degreeCelsius} and according to the manufacturer's instructions. Briefly, retronectin solution was
blocked the next day using \gls{bsa}. Prior to transduction, lentiviral adsorbed overnight at \SI{4}{\degreeCelsius} and blocked the next day using
supernatant was spinoculated at \SI{2000}{\gforce} for \SI{2}{\hour} at \gls{bsa}. Prior to transduction, lentiviral supernatant was spinoculated at
\SI{4}{\degreeCelsius}. T cells were activated in 96 well plates using beads or \SI{2000}{\gforce} for \SI{2}{\hour} at \SI{4}{\degreeCelsius}. T cells were
DMSs for \SI{24}{\hour}, and then cells and beads/\glspl{dms} were transferred activated in 96 well plates using beads or \glspl{dms} for \SI{24}{\hour}, and
onto lentiviral vector coated plates and incubated for another \SI{24}{\hour}. then cells and beads/\glspl{dms} were transferred onto lentiviral vector coated
Cells and beads/\glspl{dms} were removed from the retronectin plates using plates and incubated for another \SI{24}{\hour}. Cells and beads/\glspl{dms}
vigorous pipetting and transferred to another 96 well plate wherein expansion were removed from the retronectin plates using vigorous pipetting and
continued. transferred to another 96 well plate wherein expansion continued.
% TODO add statistics section (anova, regression, and causal inference) \subsection{statistical analysis}
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, the significance of main and interaction effects
was determined using the car library in R.
% TODO not all of this stuff applied to my regressions
For least-squares linear regression, statistical significance was evaluated the
\inlinecode{lm} function in R. Stepwise regression models were obtained using
the \inlinecode{stepAIC} function from the \inlinecode{MASS} package with
forward and reverse stepping. 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). Statistical significance was evaluated at $\upalpha$ =
0.05.
% TODO add meta-analysis section
\section{results} \section{results}
\section{discussion} \section{discussion}