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

ENH update aim 1 methods section

This commit is contained in:
Nathan Dwarshuis 2021-07-23 11:53:15 -04:00
parent 52d9fc8e16
commit 1a03a47131
2 changed files with 156 additions and 346 deletions
Show Stats Download Patch File Download Diff File Expand all files Collapse all files

230
tex/thesis.bbl
View File

@ -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}

272
tex/thesis.tex
View File

@ -65,6 +65,10 @@
\newacronym{macs}{MACS}{magnetic activated cell sorting}
\newacronym{aopi}{AO/PI}{acridine orange/propidium iodide}
\newacronym{igg}{IgG}{immunoglobulin G}
\newacronym{pe}{PE}{phycoerythrin}
\newacronym{ptnl}{PTN-L}{Protein L}
\newacronym{af647}{AF647}{Alexa Fluor 647}
\newacronym{anova}{ANOVA}{analysis of variance}
\newacronym{crispr}{CRISPR}{clustered regularly interspaced short
palindromic repeats}
@ -105,17 +109,24 @@
}
\newcommand{\invivo}{\textit{in vivo}}
\newcommand{\invitro}{\textit{in vitro}}
\newcommand{\exvivo}{\textit{ex vivo}}
\newcommand{\cd}[1]{CD{#1}}
\newcommand{\anti}[1]{anti-{#1}}
\newcommand{\anticd}[1]{\anti{\cd{#1}}}
\newcommand{\acd}[1]{\anti{\cd{#1}}}
\newcommand{\cdp}[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
@ -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
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 \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,
Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers
(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
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
throughout the bioprocess industry for adherent cultures such as stem cells and
\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}.
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
nanobead (Miltenyi TransACT), but also in soluble forms in the case of antibody
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
compared to state-of-the-art systems.
Here we propose a method using microcarriers functionalized with anti-CD3 and
anti-CD28 \glspl{mab} for use in T cell expansion cultures. Microcarriers have
Here we propose a method using microcarriers functionalized with \acd{3} and
\acd{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 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
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
microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} will
provide superior expansion and memory phenotype compared to state-of-the-art
bead-based T cell expansion technology.
@ -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
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).
volume ratio. \product{\Gls{snb}}{\thermo}{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
@ -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
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.
To coat with \gls{stp}, \SI{40}{\ug\per\mL} \product{\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
\catnum{\bl}{317320} and CD28 \catnum{\bl}{302904} were combined in a 1:1 mass
ratio and added to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along
with the \glspl{mab}, sterile \product{\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 \gls{mab} concentration using an
\product{\anti{\gls{igg}} \gls{elisa} kit}{Abcam}{157719}. Fully functionalized
\glspl{dms} were washed in sterile \gls{pbs} analogous to the previous washing
step to remove excess \gls{stp}. 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
@ -699,21 +710,21 @@ was then manually counted to obtain a concentration. Surface area for
\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}.
Biotin was quantified using the \product{\gls{haba} assay}{Sigma}{H2153-1VL}. In
the case of quantifying \gls{snb} prior to adding it to the microcarriers, the
sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar} NaOH
and allowed to react for \SI{1}{\minute} in order to prevent the reactive ester
linkages from binding to the avidin proteins in the \gls{haba}/avidin premix.
All quantifications of \gls{haba} were performed on an Eppendorf D30
Spectrophotometer using \product{\SI{70}{\ul} cuvettes}{BrandTech}{759200}. The
extinction coefficient at \SI{500}{\nm} for \gls{haba}/avidin was assumed to be
\SI{34000}{\per\cm\per\molar}.
\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{stp} binding to the carriers was quantified indirectly using a
\product{\gls{bca} kit }{\thermo}{23227} according to the manufacturer’s
instructions, with the exception that the standard curve was made with known
concentrations of purified \gls{stp} instead of \gls{bsa}. Absorbance at
\SI{592}{\nm} was quantified using a 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
@ -721,103 +732,132 @@ 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.
quantified indirectly using \product{FITC-biotin}{\thermo}{B10570}.
Briefly, \SI{400}{\pmol\per\ml} FITC-biotin were added to \gls{stp}-coated
carriers and allowed to react for \SI{20}{\minute} at room temperature under
constant agitation. The supernatant was quantified against a standard curve of
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.
adding biotin-FITC to the \gls{stp}-coated \glspl{dms}, resuspending in
\SI{1}{\percent} agarose gel, and imaging on a \product{lightsheet
microscope}{Zeiss}{Z.1}. \Gls{mab} binding was verified visually by first
staining with \product{\anti{gls{igg}}-FITC}{\bl}{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.
% TODO verify countess product number
Cryopreserved primary human T cells were either obtained as sorted
\product{\cdp{3} T cells}{Astarte Biotech}{1017} or isolated from
\product{\glspl{pbmc}}{Zenbio}{SER-PBMC} using a negative selection
\product{\cdp{3} \gls{macs} kit}{\miltenyi}{130-096-535}. T cells were activated
using \glspl{dms} or \product{\SI{3.5}{\um} CD3/CD28 magnetic
beads}{\miltenyi}{130-091-441}. In the case of beads, T cells were activated
at the manufacturer recommended cell:bead ratio of 2:1. In the case of
\glspl{dms}, cells were activated using \SI{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. Serum-free media was either
\product{OpTmizer}{\thermo}{A1048501} or
\product{TexMACS}{\miltenyi}{170-076-307} supplemented with
\SIrange{100}{400}{\IU\per\ml} \product{\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
\product{trypan blue}{\thermo}{T10282} or \product{\gls{aopi}}{Nexcelom
Bioscience}{CS2-0106-5} and a \product{Countess Automated Cell Counter}{Thermo
Fisher}{Countess 3 FL}. Media was added to cultures every \SIrange{2}{3}{\day}
depending on media color or a \SI{300}{\mg\per\deci\liter} minimum glucose
threshold. Media glucose was measured using a \product{GlucCell glucose
meter}{Chemglass}{CLS-1322-02}.
% this belongs in aim 2
% TODO 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.
Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul}
\product{\gls{stp}-\gls{pe}}{\bl}{405204} and \SI{2}{ul}
\product{\acd{45}-\gls{af647}}{\bl}{368538}, incubating for \SI{1}{\hour}, and
imaging on a spinning disk confocal microscope.
\subsection{chemotaxis assay}
% TODO not sure about the transwell product number
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.
described\cite{Hromas1997}. Briefly, \SI{3e5}{\cell} were loaded into a
\product{transwell plate with \SI{5}{\um} pore size}{Corning}{3421} with the
basolateral chamber loaded with \SI{600}{\ul} media and 0, 250, or
\SI{1000}{\ng\per\mL} \product{CCL21}{Peprotech}{250-13}. The plate was
incubated for \SI{4}{\hour} after loading, and the basolateral chamber of each
transwell was quantified for total cells using \product{countbright
beads}{\thermo}{C36950}. The final readout was normalized using the
\SI{0}{\ng\per\mL} concentration as background.
\subsection{degranulation assay}
Cytotoxicity of expanded 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.
Cytotoxicity of expanded \gls{car} T cells was assessed using a degranulation
assay as previously described\cite{Schmoldt1975}. Briefly, \num{3e5} T cells
were incubated with \num{1.5e5} target cells consisting of either \product{K562
wild type cells}{ATCC}{CCL-243} or CD19- expressing K562 cells transformed
with \gls{crispr} (kindly provided by Dr.\ Yvonne Chen, UCLA)\cite{Zah2016}.
Cells were seeded in a flat bottom 96 well plate with \SI{1}{\ug\per\ml}
\product{\acd{49d}}{eBioscience}{16-0499-81}, \SI{2}{\micro\molar} \product{monensin}{eBioscience}{
00-4505-51}, and \SI{1}{\ug\per\ml} \product{\acd{28}}{eBioscience}{302914} (all
functional grade \glspl{mab}) with \SI{250}{\ul} total volume. After
\SI{4}{\hour} incubation at \SI{37}{\degreeCelsius}, cells were stained for CD3,
CD4, and CD107a and analyzed on a BD LSR Fortessa. Readout was calculated as the
percent \cdp{107a} cells of the total \cdp{8} fraction.
\subsection{car expression}
% 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).
\gls{car} expression was quantified as previously described\cite{Zheng2012}.
Briefly, cells were washed once and stained with \product{biotinylated
\gls{ptnl}}{\thermo}{29997}. After a subsequent wash, cells were stained with
\product{\gls{pe}-\gls{stp}}{\bl}{405204}, washed again, and analyzed on a
BD Accuri. Readout was percent \gls{pe}+ cells as compared to secondary controls
(\gls{pe}-\gls{stp} with no \gls{ptnl}).
\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.
The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$
promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a
\product{FUGW}{Addgene}{14883} kindly provided by the Emory Viral Vector Core.
Lentiviral vectors were synthesized by the Emory Viral Vector Core or the
Cincinnati Children's Hospital Medical Center Viral Vector Core. To transduce
primary human T cells, \product{retronectin}{Takara}{T100A} was coated onto
non-TC treated 96 well plates and used to immobilize lentiviral vector particles
according to the manufacturer's instructions. Briefly, retronectin solution was
adsorbed overnight at \SI{4}{\degreeCelsius} and blocked the next day using
\gls{bsa}. Prior to transduction, lentiviral supernatant was spinoculated at
\SI{2000}{\gforce} for \SI{2}{\hour} at \SI{4}{\degreeCelsius}. T cells were
activated in 96 well plates using beads or \glspl{dms} for \SI{24}{\hour}, and
then cells and beads/\glspl{dms} were transferred onto lentiviral vector coated
plates and incubated for another \SI{24}{\hour}. Cells and beads/\glspl{dms}
were removed from the retronectin plates using vigorous pipetting and
transferred to another 96 well plate wherein expansion continued.
% 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{discussion}