ENH proof aim 3
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tex/thesis.tex
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tex/thesis.tex
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@ -380,6 +380,7 @@
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\newcommand{\inlinecode}{\texttt}
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\newcommand{\inlinecode}{\texttt}
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\newcommand{\subcap}[2]{\subref{#1}) #2}
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\newcommand{\subcap}[2]{\subref{#1}) #2}
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\newcommand{\sigkey}{Significance test key: *p<0.1; **p < 0.05; ***p<0.01}
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\newcommand{\sigkey}{Significance test key: *p<0.1; **p < 0.05; ***p<0.01}
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\newcommand{\nVI}{NALM-6}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% ditto for environments
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% ditto for environments
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@ -587,7 +588,7 @@ The goal of this dissertation was to develop a microcarrier-based \gls{dms} T
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cell expansion system and determine biologically-meaningful \glspl{cqa} and
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cell expansion system and determine biologically-meaningful \glspl{cqa} and
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\glspl{cpp} that could be used to optimize for highly-potent T cells. In
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\glspl{cpp} that could be used to optimize for highly-potent T cells. In
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\cref{aim1}, we developed and characterized the \gls{dms} system, including
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\cref{aim1}, we developed and characterized the \gls{dms} system, including
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quality control steps. We also demonstrated the feasibility of expanding
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\gls{qc} steps. We also demonstrated the feasibility of expanding
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high-quality T cells. In \cref{aim2a,aim2b}, we used \gls{doe} methodology to
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high-quality T cells. In \cref{aim2a,aim2b}, we used \gls{doe} methodology to
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optimize the \gls{dms} platform, and we developed a computational pipeline to
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optimize the \gls{dms} platform, and we developed a computational pipeline to
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identify and model the effects of measurable \glspl{cqa} and \glspl{cpp} on the
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identify and model the effects of measurable \glspl{cqa} and \glspl{cpp} on the
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@ -687,7 +688,7 @@ The specific aims of this dissertation are outlined in
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mimics key components of the lymph nodes}
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mimics key components of the lymph nodes}
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In this first aim, we demonstrated the process for manufacturing \glspl{dms},
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In this first aim, we demonstrated the process for manufacturing \glspl{dms},
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including quality control steps that are necessary for translation of this
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including \gls{qc} steps that are necessary for translation of this
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platform into a scalable manufacturing setting. We also demonstrated that the
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platform into a scalable manufacturing setting. We also demonstrated that the
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\gls{dms} platform leads to higher overall expansion of T cells and higher
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\gls{dms} platform leads to higher overall expansion of T cells and higher
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overall fractions of potent memory and CD4+ subtypes desired for T cell
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overall fractions of potent memory and CD4+ subtypes desired for T cell
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@ -1038,8 +1039,8 @@ Matrigel\cite{Rio2018} or 3d-printed lattices\cite{Delalat2017}, ellipsoid
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beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms}
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beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms}
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beads\cite{Lambert2017} that respectively recapitulate the cellular membrane,
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beads\cite{Lambert2017} that respectively recapitulate the cellular membrane,
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large interfacial contact area, 3D-structure, or soft surfaces T cells normally
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large interfacial contact area, 3D-structure, or soft surfaces T cells normally
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experience \textit{in vivo}. None of these have been shown to expand high
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experience \invivo{}. None of these have been shown to expand high quality T
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quality T cells as outlined in \cref{sec:background_quality}.
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cells as outlined in \cref{sec:background_quality}.
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\subsection{Microcarriers in Bioprocessing}
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\subsection{Microcarriers in Bioprocessing}
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@ -1672,7 +1673,7 @@ to secondary controls (\gls{pe}-\gls{stp} with no \gls{ptnl}).
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was added to tubes analogously to \gls{ptnl} and incubated for \SI{45}{\minute}
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was added to tubes analogously to \gls{ptnl} and incubated for \SI{45}{\minute}
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prior to analyzing on a \bd{} Accuri
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prior to analyzing on a \bd{} Accuri
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\subsection{CAR Plasmid and Lentiviral Transduction}
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\subsection{CAR Plasmid and Lentiviral Transduction}\label{sec:transduction}
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The anti-CD19-CD8-CD137-CD3$\upzeta$ \gls{car} sequence with the EF1$\upalpha$
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The anti-CD19-CD8-CD137-CD3$\upzeta$ \gls{car} sequence with the EF1$\upalpha$
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promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a
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promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a
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@ -4115,27 +4116,6 @@ results on expansion and memory phenotype. Essentially this would turn the
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\glspl{dms} into stromal cells that present \il{15}, as seen to be important in
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\glspl{dms} into stromal cells that present \il{15}, as seen to be important in
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the early work with \il{15} in mice\cite{Lodolce1998}.
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the early work with \il{15} in mice\cite{Lodolce1998}.
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% DISCUSSION not sure if this belongs here, although it might make sense to offer
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% alternative explanations of why the DMSs "work" given this negative data
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% Second, there is evidence that providing a larger
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% contact area for T cell activation provides greater stimulation16,43; the DMSs
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% have a rougher interface than the 5 µm magnetic beads, and thus could facilitate
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% these larger contact areas. Third, the DMSs may allow the T cells to cluster
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% more densely compared to beads, as evidenced by the large clusters on the
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% outside of the DMSs (Figure 1f) as well as the significant fraction of DMSs
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% found within their interiors (Supplemental Figure 2a and b). This may alter the
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% local cytokine environment and trigger different signaling pathways.
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% Particularly, IL15 and IL21 are secreted by T cells and known to drive memory
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% phenotype44–46. We noted that the IL15 and IL21 concentration was higher in a
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% majority of samples when comparing beads and DMSs across multiple timepoints
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% (Supplemental Figure 18) in addition to many other cytokines. IL15 and IL21 are
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% added exogenously to T cell cultures to enhance memory frequency,45,47 and our
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% data here suggest that the DMSs are better at naturally producing these
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% cytokines and limiting this need. Furthermore, IL15 unique signals in a trans
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% manner in which IL15 is presented on IL15R to neighboring cells48. The higher
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% cell density in the DMS cultures would lead to more of these trans interactions,
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% and therefore upregulate the IL15 pathway and lead to more memory T cells.
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\chapter{AIM 3}\label{aim3}
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\chapter{AIM 3}\label{aim3}
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\section{Introduction}
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\section{Introduction}
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@ -4156,44 +4136,47 @@ lower-differentiated T cells with higher potency\cite{Ghassemi2018}.
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\section{Methods}
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\section{Methods}
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\subsection{CD19-CAR T Cell Generation}
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\subsection{T Cell Culture}
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\subsection{T Cell Culture}
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T cells were grown as described in \cref{sec:tcellculture}.
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T cells were grown as described in \cref{sec:tcellculture}.
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\subsection{CD19-CAR T Cell Generation}
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T cells were grown as described in \cref{sec:transduction}.
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\subsection{\Invivo{} Therapeutic Efficacy in NSG Mice Model}
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\subsection{\Invivo{} Therapeutic Efficacy in NSG Mice Model}
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% METHOD describe how the luciferase cells were generated (eg the kwong lab)
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% METHOD describe how the luciferase cells were generated (eg the kwong lab)
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% METHOD use actual product numbers for mice
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% METHOD use actual product numbers for mice
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All mice in this study were male \gls{nsg} mice from Jackson Laboratories. At
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All mice in this study were male \gls{nsg} mice from Jackson Laboratories. At
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day 0 (\SI{-7}{\day} relative to T cell injection), 1e6 firefly
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day 0 (\SI{-7}{\day} relative to T cell injection), \num{1e6} firefly
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luciferase-expressing \product{Nalm-6 cells}{ATCC}{CRL-3273} suspended in
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luciferase-expressing\footnote{luciferase transduction was performed and
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ice-cold \gls{pbs} were injected via tail vein into each mouse. At day 7, saline
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verified by Ian Miller in the Kwong Lab at Georgia Tech} \product{\nVI{}
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or \gls{car} T cells at the indicated doses from either bead or
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cells}{ATCC}{CRL-3273} suspended in ice-cold \gls{pbs} were injected via tail
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\gls{dms}-expanded T cell cultures (for \SI{14}{\day}) were injected into each
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vein into each mouse. At day 7, saline or \gls{car} T cells at the indicated
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mouse via tail vein. Tumor burden was quantified longitudinally via an
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doses from either bead or \gls{dms}-expanded T cell cultures (for \SI{14}{\day})
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\gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug} luciferin at
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were injected into each mouse via tail vein. Tumor burden was quantified
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\SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally under isoflurane
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longitudinally via an \gls{ivis} Spectrum (Perkin Elmer). Briefly, \SI{200}{\ug}
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anesthesia into each mouse and allowed to circulate for at least
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luciferin at \SI{15}{\mg\per\ml} in \gls{pbs} was injected intraperitoneally
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\SI{10}{\minute} before imaging. Mice were anesthetized again and imaged using
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under isoflurane anesthesia into each mouse and allowed to circulate for at
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the \gls{ivis}. Mice from each treatment group/dose were anesthetized, injected,
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least \SI{10}{\minute} before imaging. Mice were anesthetized again and imaged
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and imaged together, and exposure time of the \gls{ivis} was limited to avoid
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using the \gls{ivis}. Mice from each treatment group/dose were anesthetized,
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saturation based on the signal from the saline group. \gls{ivis} images were
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injected, and imaged together; exposure time of the \gls{ivis} was limited to
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processed by normalizing them to common minimum and maximum photon counts and
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avoid saturation based on the signal from the saline group. \gls{ivis} images
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total flux was estimated in terms of photons/second. Endpoint for each mouse was
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were scaled to common minimum and maximum photon counts. Endpoint for each mouse
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determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis,
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was determined by \gls{iacuc} euthanasia criteria (hunched back, paralysis,
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blindness, lethargy, and weight loss). Mice were euthanized according to these
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blindness, lethargy, and weight loss). Mice were euthanized according to these
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endpoint criteria using carbon dioxide asphyxiation.
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endpoint criteria using carbon dioxide asphyxiation.
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\subsection{Statistics}
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\subsection{Statistics}
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For the \invivo{} model, the survival curves were created and statistically
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Survival curves were created and statistically analyzed using GraphPad Prism
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analyzed using GraphPad Prism using the Mantel-Cox test to assess significance
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using the Mantel-Cox test to assess significance between survival groups.
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between survival groups.
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\section{Results}
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\section{Results}
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\subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity}
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\begin{figure*}[ht!]
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\begin{figure*}[ht!]
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\begingroup
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\begingroup
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@ -4212,7 +4195,6 @@ between survival groups.
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\input{../tables/mouse_dose_car.tex}
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\input{../tables/mouse_dose_car.tex}
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\end{table}
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\end{table}
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\subsection{DMSs Lead to Greater \invivo{} Anti-Tumor Activity}
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\begin{figure*}[ht!]
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\begin{figure*}[ht!]
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\begingroup
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\begingroup
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@ -4248,7 +4230,7 @@ between survival groups.
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\caption[Mouse Dosing IVIS and Survival Results]
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\caption[Mouse Dosing IVIS and Survival Results]
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{T cells expanded with \glspl{dms} confer greater anti-tumor potency \invivo{}
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{T cells expanded with \glspl{dms} confer greater anti-tumor potency \invivo{}
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even at lower doses.
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even at lower doses.
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\subcap{fig:mouse_dosing_ivis_images}{IVIS images of Nalm-6 tumor-bearing
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\subcap{fig:mouse_dosing_ivis_images}{IVIS images of \nVI{} tumor-bearing
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\gls{nsg} mice injected with varying doses of T cells}
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\gls{nsg} mice injected with varying doses of T cells}
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\subcap{fig:mouse_dosing_ivis_plots}{Plots showing quantified photon counts
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\subcap{fig:mouse_dosing_ivis_plots}{Plots showing quantified photon counts
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of the results from (\subref{fig:mouse_dosing_ivis_plots}).}
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of the results from (\subref{fig:mouse_dosing_ivis_plots}).}
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@ -4266,11 +4248,11 @@ between survival groups.
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We asked if the higher memory/naive phenotype and more balanced CD4/CD8 ratio of
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We asked if the higher memory/naive phenotype and more balanced CD4/CD8 ratio of
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our \gls{dms}-expanded \gls{car} T cells would lead to better anti-tumor potency
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our \gls{dms}-expanded \gls{car} T cells would lead to better anti-tumor potency
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in vivo compared to bead-expanded \gls{car} T cells. We also asked if this
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\invivo{} compared to bead-expanded \gls{car} T cells. We also asked if this
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superior anti-tumor potency would hold true at lower doses of \gls{car}
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superior anti-tumor potency would hold true at lower doses of \gls{car}
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expressing T cells in the DMS group vs the bead group. To test this, we used a
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expressing T cells in the DMS group vs the bead group. To test this, we used a
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human xenograft model of B cell \gls{all} by intravenously injecting \gls{nsg}
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human xenograft model of B cell \gls{all} by intravenously injecting \gls{nsg}
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mice with \num{1e6} Nalm-6 tumor cells expression firefly
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mice with \num{1e6} \nVI{} tumor cells expressing firefly
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luciferase\cite{Fraietta2018}. After \SI{7}{\day} of tumor cell growth
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luciferase\cite{Fraietta2018}. After \SI{7}{\day} of tumor cell growth
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(\cref{fig:mouse_dosing_overview}), we intravenously injected saline or three
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(\cref{fig:mouse_dosing_overview}), we intravenously injected saline or three
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doses (high, medium, and low) of \gls{car} T cells from either bead or \gls{dms}
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doses (high, medium, and low) of \gls{car} T cells from either bead or \gls{dms}
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@ -4280,57 +4262,55 @@ groups using the \gls{ptnl} assay (\cref{tab:mouse_dosing_results}).
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Before injecting the T cells into the mice, we quantified their phenotype and
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Before injecting the T cells into the mice, we quantified their phenotype and
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growth. We observed that for this expansion, the bead and \gls{dms} T cells
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growth. We observed that for this expansion, the bead and \gls{dms} T cells
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produced similar numbers of \ptmem{} T cells, and the beads even had a higher
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produced similar numbers of \ptmem{} T cells, and the beads even had a higher
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fraction of CD45RA, which is present on lower-differentiated \glspl{tn} and
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fraction of \cdp{45RA} cells, which is present on lower-differentiated
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\glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the \pthp{} of
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\glspl{tn} and \glspl{tscm} (\cref{fig:mouse_dosing_qc_mem}). However, the
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the final product was higher in \gls{dms} (\cref{fig:mouse_dosing_qc_cd4}). The
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\pthp{} of the final product was higher in \gls{dms}
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\gls{dms} T cells also expanded more robustly than the beads
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(\cref{fig:mouse_dosing_qc_cd4}). The \gls{dms} T cells also expanded more
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(\cref{fig:mouse_dosing_qc_growth}).
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robustly than the beads (\cref{fig:mouse_dosing_qc_growth}).
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In the Nalm-6/\gls{nsg} xenograft model, we observed lower tumor burden and
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In the \nVI{}/\gls{nsg} xenograft model, bead and \gls{dms}-treated mice at all
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significantly longer survival of bead and \gls{dms}-treated mice at all doses
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doses had lower tumor burden and significantly longer survival compared to the
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compared to the saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at
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saline groups (\cref{fig:mouse_dosing_ivis}). Importantly, at each dose the
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each dose we observed that the \gls{dms}-treated mice had much lower tumor
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\gls{dms}-treated mice had much lower tumor burden and significantly higher
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burden and significantly higher survival than their bead-treated counterparts
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survival than their bead-treated counterparts
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(\cref{fig:mouse_dosing_ivis_survival}). When factoring the percentage T cells
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(\cref{fig:mouse_dosing_ivis_survival}). When factoring the percentage T cells
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in each dose that expressed the \gls{car}, we note that survival of the low
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in each dose that expressed the \gls{car}, survival of the low \gls{dms} dose
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\gls{dms} dose (which had similar total \gls{car} T cells compared to the bead
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(which had similar total \gls{car} T cells compared to the bead medium dose and
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medium dose and less than the bead high dose) is significantly higher than that
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less than the bead high dose) was significantly higher than that of both the
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of both the bead medium dose and the bead high dose
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bead medium dose and the bead high dose
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(\cref{fig:mouse_dosing_ivis_survival_comp}). Overall, the Kaplan-Meier survival
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(\cref{fig:mouse_dosing_ivis_survival_comp}). Overall, the Kaplan-Meier survival
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of Nalm-6 tumor bearing \gls{nsg} mice shown in the
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of \nVI{} tumor bearing \gls{nsg} mice shown in the
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\cref{fig:mouse_dosing_ivis_survival} was up to day 40 as reported
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\cref{fig:mouse_dosing_ivis_survival} was up to day 40 as reported
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elsewhere\cite{Fraietta2018}. However, we also included a Kaplan-Meier figure up
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elsewhere\cite{Fraietta2018}. However, most of the mice euthanized from day 40
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to day 46 (\cref{fig:mouse_dosing_ivis_survival_full}) where most of the mice
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through day 46 from \gls{dms} groups showed no or very small fragment of spleen
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euthanized from day 40 through day 46 from \gls{dms} groups showed no or very
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which was due to \gls{gvhd} responses
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small fragment of spleen which was due to \gls{gvhd} responses. Similar
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(\cref{fig:mouse_dosing_ivis_survival_full}). Similar \gls{gvhd} responses
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\gls{gvhd} responses were reported earlier in \gls{nsg} mice where the mice
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\SIrange{40}{50}{\day} after injection have been reported by others in \gls{nsg}
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injected with human \gls{pbmc} exhibited acute \gls{gvhd} between
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mice injected with human \gls{pbmc}\cite{Ali2012}. Both survival analyses (up to
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\SIrange{40}{50}{\day} post intravenous injection\cite{Ali2012}. Notably, both
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day 40 in \cref{fig:mouse_dosing_ivis_survival} and up to day 46 in
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survival analyses (up to day 40 in \cref{fig:mouse_dosing_ivis_survival} and up
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\cref{fig:mouse_dosing_ivis_survival_full}) confirmed that \gls{dms}-expanded
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to day 46 in \cref{fig:mouse_dosing_ivis_survival_full}) confirmed that
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groups outperformed bead-expanded groups in terms of prolonging survival of
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\gls{dms}-expanded groups outperformed bead-expanded groups in terms of
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\nVI{} tumor challenged \gls{nsg} mice.
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prolonging survival of Nalm-6 tumor challenged \gls{nsg} mice.
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Together, these data suggested that \glspl{dms} produce T cells that are not
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Together, these data suggested that \glspl{dms} produce T cells that are not
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only more potent that bead-expanded T cells (even when accounting for
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only more potent that bead-expanded T cells (even when accounting for
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differences in \gls{car} expression) but also showed that \gls{dms} expanded T
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differences in \gls{car} expression) but also showed that \gls{dms} expanded T
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cells are effective at lower doses. Given the quality control data of the T
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cells are effective at lower doses. Given the \gls{qc} data of T cells prior to
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cells prior to injecting into the mice, it seems that this advantage is either
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injection, it seems that this advantage for \gls{dms} groups was either due to
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due to the higher \pthp{} or the overall fitness of the T cells given the higher
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higher \pthp{} or greater overall fitness (implied by higher fold change)
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expansion in the case of \gls{dms}
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(\cref{fig:mouse_dosing_qc_cd4,fig:mouse_dosing_qc_growth}). It was likely not
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(\cref{fig:mouse_dosing_qc_cd4,fig:mouse_dosing_qc_growth}). It was likely not
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due to the memory phenotype given that it was actually slightly higher in the
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due to memory phenotype given that this was actually slightly higher for the
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case of beads (\cref{fig:mouse_dosing_qc_mem}).
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bead culture (\cref{fig:mouse_dosing_qc_mem}).
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\subsection{Beads and DMSs Perform Similarly at Earlier Timepoints}
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\subsection{Beads and DMSs Perform Similarly at Earlier Timepoints}
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We then asked how T cells harvested using either beads or \gls{dms} performed
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We then asked how T cells activated using beads or \gls{dms} performed when
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when harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same
|
harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same
|
||||||
experiments as described in \cref{fig:mouse_dosing_overview} with the
|
experiments as described in \cref{fig:mouse_dosing_overview} with the
|
||||||
modification that T cells were only grown and harvested after \SI{6}{\day},
|
modification that T cells were only expanded and harvested after \SI{6}{\day},
|
||||||
\SI{10}{\day}, or \SI{14}{\day} of expansion
|
\SI{10}{\day}, or \SI{14}{\day} of expansion
|
||||||
(\cref{fig:mouse_timecourse_overview}). T cells were frozen after harvest, and
|
(\cref{fig:mouse_timecourse_overview}). T cells were frozen after harvest, and
|
||||||
all timepoints were thawed at the same time prior to injection. The dose of T
|
all timepoints were thawed simultaneously prior to injection. The dose of T
|
||||||
cells injected was \num{1.25e6} cells per mouse (the same as the high dose in
|
cells injected was \num{1.25e6} cells per mouse (the same as the high dose in
|
||||||
the first experiment). All other characteristics of the experiment were the
|
the first experiment). All other characteristics of the experiment were the
|
||||||
same.
|
same.
|
||||||
|
@ -4348,20 +4328,19 @@ same.
|
||||||
\label{fig:mouse_timecourse_overview}
|
\label{fig:mouse_timecourse_overview}
|
||||||
\end{figure*}
|
\end{figure*}
|
||||||
|
|
||||||
As was the case with the first \invivo{} experiment, T cells activated with
|
As was the case with the first \invivo{} experiment, \gls{dms} cultures expanded
|
||||||
\glspl{dms} expanded much more efficiently compared to those expanded with beads
|
much more efficiently than bead cultures
|
||||||
(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} of T
|
(\cref{fig:mouse_timecourse_qc_growth}). When we quantified the \ptcarp{} at
|
||||||
cells harvested at each timepoint, we noted that the bead group had much higher
|
each timepoint, the bead group had much higher \ptcar{} expression at earlier
|
||||||
\ptcar{} expression at earlier timpoints compared to \gls{dms}, while they
|
timpoints compared to \gls{dms}, while they equalized at later timepoints
|
||||||
equalized at later timepoints (\cref{fig:mouse_timecourse_qc_car}). In addition,
|
(\cref{fig:mouse_timecourse_qc_car}). In addition, overall \ptcar{} expression
|
||||||
overall \ptcar{} expression decreased at later timepoints, indicating that
|
decreased at later timepoints, indicating that transduced cells either grew
|
||||||
\gls{car} transduced T cells either grow slower or died faster compared to
|
slower or died faster compared to untransduced cells. The \pthp{} was higher
|
||||||
untransduced cells. The \pthp{} of the harvested T cells was higher overall in
|
overall in \gls{dms} groups but decreased with increasing timepoints
|
||||||
\gls{dms} expanded T cells but decreased with increasing timepoints
|
(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6 between
|
||||||
(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6
|
bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{} at day 14
|
||||||
between bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{}
|
despite the overall \ptmemp{} decreasing with time
|
||||||
at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere
|
(\cref{fig:mouse_timecourse_qc_mem}).
|
||||||
(\cref{fig:mouse_timecourse_qc_mem})\cite{Ghassemi2018}.
|
|
||||||
|
|
||||||
\begin{figure*}[ht!]
|
\begin{figure*}[ht!]
|
||||||
\begingroup
|
\begingroup
|
||||||
|
@ -4386,20 +4365,19 @@ at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere
|
||||||
\label{fig:mouse_timecourse_qc}
|
\label{fig:mouse_timecourse_qc}
|
||||||
\end{figure*}
|
\end{figure*}
|
||||||
|
|
||||||
We analyzed the tumor burden using \gls{ivis} which showed that mice that
|
Analyzing the tumor burden using \gls{ivis} showed that mice who received T
|
||||||
received T cells from any group performed better than those that received only
|
cells from any group had less tumor than those that received only saline
|
||||||
saline (\cref{fig:mouse_timecourse_ivis}). Note that unlike the previous
|
(\cref{fig:mouse_timecourse_ivis}). Unlike the previous experiment, most mice
|
||||||
experiment, many of the mice survived until day 40 at which point \gls{gvhd}
|
survived until day 40 after which \gls{gvhd} began to take effect (upon
|
||||||
began to take effect (after euthanizing the mice at day 42, most had small or no
|
euthanization at day 42, most had little or no spleen). When comparing bead and
|
||||||
spleen). When comparing bead and \gls{dms} groups, the \gls{dms} T cells still
|
\gls{dms} groups, the \gls{dms} groups had lower tumor than the bead group, at
|
||||||
seemed superior to the bead group, at least initially (note that in this case
|
least initially (note that in this experiment they had similar numbers of
|
||||||
they had similar numbers of \ptcar{} cells). At day 6, both \gls{dms} and bead
|
\ptcar{} cells). For day 6 groups, both treatments seemed to eradicate the tumor
|
||||||
groups seemed to eradicate the tumor initially, after which it came back after
|
initially, then it came back after \SI{21}{\day} for the beads and \SI{28}{\day}
|
||||||
day 21 for the bead and day 28 for the \gls{dms} group. The day 10 groups
|
for \glspl{dms}. The day 10 groups performed somewhere in between, where they
|
||||||
performed somewhere in between, where they increased linearly unlike the day 6
|
increased linearly unlike the day 6 groups but not as quickly as the day 14
|
||||||
groups but not as quickly as the day 14 groups. In the case of the \gls{dms} day
|
groups. In the case of the \gls{dms} day 10 group, a few mice actually had less
|
||||||
10 group, it also appeared like a few mice actually performed better than all
|
tumor burden overall than all other groups.
|
||||||
other groups in regard to the final tumor burden.
|
|
||||||
|
|
||||||
\begin{figure*}[ht!]
|
\begin{figure*}[ht!]
|
||||||
\begingroup
|
\begingroup
|
||||||
|
@ -4433,85 +4411,76 @@ other groups in regard to the final tumor burden.
|
||||||
|
|
||||||
\endgroup
|
\endgroup
|
||||||
\caption[Mouse Summary]
|
\caption[Mouse Summary]
|
||||||
{Summary of cells injected into mice during for
|
{Summary of T cells injected into mice for the
|
||||||
\subcap{fig:mouse_summary_1}{the first mouse experiment} and
|
\subcap{fig:mouse_summary_1}{first} and \subcap{fig:mouse_summary_2}{second}
|
||||||
\subcap{fig:mouse_summary_2}{the second mouse experiment}. The y axis
|
experiments. The y-axis maximum is set to the maximum cell number
|
||||||
maximum is set to the maximum number of cells injected between both
|
injected between both experiments (\num{1.25e6}). NOTE: the \gls{car} was
|
||||||
experiments (\num{1.25e6}). Note that the \gls{car} was quantified using a
|
quantified using a separate panel from the other markers. }
|
||||||
separate panel than the rest of the markers.
|
|
||||||
}
|
|
||||||
\label{fig:mouse_summary}
|
\label{fig:mouse_summary}
|
||||||
\end{figure*}
|
\end{figure*}
|
||||||
|
|
||||||
The total number of T cells injected for each \invivo{} experiment are shown in
|
When we tested bead- and \gls{dms}-expanded \gls{car} T cells, the latter
|
||||||
\cref{fig:mouse_summary}.
|
prolonged survival compared to the former in \nVI{} tumor challenged
|
||||||
|
(intravenously injected) \gls{nsg} mice. This held true when matching groups for
|
||||||
|
absolute \gls{car} dose. Furthermore, \gls{dms}-expanded \gls{car} T cells were
|
||||||
|
effective in clearing tumor cells as early as \SI{7}{\day} post T injection even
|
||||||
|
at low dose compared to the bead groups where tumor burden was higher than
|
||||||
|
\gls{dms} groups across all the total T cell doses tested here. These suggest
|
||||||
|
that \glspl{dms} (compared to beads) produced highly effective \gls{car} T cells
|
||||||
|
that can efficiently kill tumor cells.
|
||||||
|
|
||||||
When we tested bead and \gls{dms} expanded \gls{car} T cells, we found that the
|
When comparing total number of injected T cells with different phenotypes, the
|
||||||
\gls{dms} expanded \gls{car} T cells outperformed bead groups in prolonging
|
number of \ptmem{} (both with and without CD45RA) cells was lower in the
|
||||||
survival of Nalm-6 tumor challenged (intravenously injected) \gls{nsg} mice.
|
low-dose \gls{dms} group compared to the med-dose bead group (which had similar
|
||||||
\gls{dms} expanded CAR-T cells were very effective in clearing tumor cells as
|
numbers of \gls{car} T cells) (\cref{fig:mouse_summary_1}). This could mean
|
||||||
early as \SI{7}{\day} post \gls{car} T injection even at low total T cell dose
|
several things. First, the \ptmem{} phenotype may have nothing to do with the
|
||||||
compared to the bead groups where tumor burden was higher than \gls{dms} groups
|
results seen here, at least in this model. While this may have been the case in
|
||||||
across all the total T cell doses tested here. More interestingly, when only
|
our hands, this would contradict previous evidence suggesting that \gls{tn} and
|
||||||
\gls{car}-expressing T cell doses between bead and \gls{dms} groups were
|
\gls{tcm} cells work better in almost the same model (the only difference being
|
||||||
compared, \gls{dms} group had significantly higher survival effects over similar
|
Raji cells in place of \nVI{} cells, both of which express
|
||||||
or higher CAR expression T cell doses from bead group. All these results suggest
|
CD19)\cite{Sommermeyer2015}. Second, the distribution of \gls{car} T cells
|
||||||
that the T cells in \gls{dms} groups (compared to bead group) resulted in highly
|
across different subtypes of T cells was different between the \gls{dms} and
|
||||||
effective \gls{car} T cells that can efficiently kill tumor cells.
|
bead groups (with possibly higher correlation of \gls{car} expression and the
|
||||||
|
\ptmem{} phenotype). It is hard to assess this without strong assumptions as the
|
||||||
|
\gls{car} was quantified using a separate flow panel relative to the other
|
||||||
|
markers.
|
||||||
|
|
||||||
When comparing the total number of T cells of different phenotypes, we observed
|
We can make a similar observation for the number of \pth{} T cells injected
|
||||||
that when comparing low-dose \gls{dms} group to the mid- bead groups (which had
|
|
||||||
similar numbers of \gls{car} T cells), the number of \ptmem{} (both with and
|
|
||||||
without CD45RA) T cells injected was much lower in the \gls{dms} group
|
|
||||||
(\cref{fig:mouse_summary_1}). This could mean several things. First, the
|
|
||||||
\ptmem{} phenotype may have nothing to do with the results seen here, at least
|
|
||||||
in this model. While this may have been the case in our hands, this would
|
|
||||||
contradict previous evidence suggesting that \gls{tn} and \gls{tcm} cells work
|
|
||||||
better in almost the same model (the only difference being Raji cells in place
|
|
||||||
of Nalm-6 cells, both of which express CD19)\cite{Sommermeyer2015}. Second, the
|
|
||||||
distribution of \gls{car} T cells across different subtypes of T cells was
|
|
||||||
different between the \gls{dms} and bead groups (with possibly higher
|
|
||||||
correlation of \gls{car} expression and the \ptmem{} phenotype). It is hard to
|
|
||||||
assess this without strong assumptions as the \gls{car} was quantified using a
|
|
||||||
separate flow panel relative to the other markers.
|
|
||||||
|
|
||||||
We can also make a similar observation for the number of \pth{} T cells injected
|
|
||||||
(\cref{fig:mouse_summary_1}). In this case, either the \pth{} phenotype doesn't
|
(\cref{fig:mouse_summary_1}). In this case, either the \pth{} phenotype doesn't
|
||||||
matter in this model (or the \ptk{} population matters much more), or the
|
matter in this model (or the \ptk{} population matters much more), or the
|
||||||
distribution of \gls{car} is different between CD4 and CD8 T cells in a manner
|
distribution of \gls{car} is different between CD4 and CD8 T cells in a manner
|
||||||
that favors the \gls{dms} group. While in a glioblastoma model and not a B-cell
|
that favors the \gls{dms} group. Previous groups have shown that \pthp{} T cells
|
||||||
\gls{all} model, previous groups have shown that \pthp{} T cells are important
|
are important for response (albeit for a glioblastoma model and not a B-cell
|
||||||
for response\cite{Wang2018}.
|
\gls{all} model)\cite{Wang2018}.
|
||||||
|
|
||||||
When testing \gls{car} T cells at earlier timepoints relative to day 14 as used
|
When testing \gls{car} T cells at earlier timepoints relative to day 14 as used
|
||||||
in the first \invivo{} experiment, we noted that none of the \gls{car}
|
in the first \invivo{} experiment, none of the \gls{car} treatments seemed to
|
||||||
treatments seemed to work as well as they did in the first experiment. However,
|
work as well as they did in the first experiment. However, the total number of
|
||||||
the total number of \gls{car} T cells was generally much lower in this second
|
\gls{car} T cells was generally much lower in this second experiment relative to
|
||||||
experiment relative to the first (\cref{fig:mouse_summary}). Only the day 6
|
the first (\cref{fig:mouse_summary}). Only the day 6 group had \gls{car} T cell
|
||||||
group had \gls{car} T cell numbers comparable to the weakest dose of bead cells
|
numbers comparable to the weakest dose of bead cells given in the first
|
||||||
given in the first experiment, and these T cells were harvested at earlier
|
experiment, and these T cells were harvested at earlier timepoints than the
|
||||||
timepoints than the first mouse experiment and thus may not be safely
|
first mouse experiment and thus are not directly comparable. Furthermore, the
|
||||||
comparable. Furthermore, the \ptcarp{} decreased over time, which suggested that
|
\ptcarp{} decreased over time, which suggested that the transduced T cells grew
|
||||||
the transduced T cells grew slower. This has been observed elsewhere and could
|
slower. This has been observed elsewhere and could be due to tonic
|
||||||
be due to tonic signaling\cite{GomesSilva2017}. The lower overall \gls{car}
|
signaling\cite{GomesSilva2017}. The lower overall \gls{car} doses may explain
|
||||||
doses may explain why at best, the tumor seemed to be in remission only
|
why at best, the tumor seemed to be in remission only temporarily. Even so, the
|
||||||
temporarily. Even so, the \gls{dms} group seemed to perform better at day 6 as
|
\gls{dms} group seemed to perform better at day 6 as it held off the tumor
|
||||||
it held off the tumor longer, and also slowed the tumor progression relative to
|
longer, and also slowed the tumor progression relative to the bead group at day
|
||||||
the bead group at day 14 (\cref{fig:mouse_timecourse_ivis_plots}).
|
14 (\cref{fig:mouse_timecourse_ivis_plots}).
|
||||||
|
|
||||||
Taken together, these data suggest that the \gls{dms} platform produces T cells
|
Taken together, these data suggest that the \gls{dms} platform produces T cells
|
||||||
that have an advantage \invivo{} over beads. While we may not know the exact
|
that have an advantage \invivo{} over beads. While we may not know the exact
|
||||||
mechanism, our data suggests that the responses are unsurprisingly influenced by
|
mechanism, our data suggests that the responses are unsurprisingly influenced by
|
||||||
the \ptcarp{} of the final product. Followup experiments would need to be
|
the \ptcarp{} of the final product. Followup experiments are needed to determine
|
||||||
performed to determine the precise phenotype responsible for these responses in
|
the precise phenotype responsible for these results.
|
||||||
our hands.
|
|
||||||
|
|
||||||
\chapter{CONCLUSIONS AND FUTURE WORK}\label{conclusions}
|
\chapter{CONCLUSIONS AND FUTURE WORK}\label{conclusions}
|
||||||
|
|
||||||
\section{Conclusions}
|
\section{Conclusions}
|
||||||
|
|
||||||
This dissertation describes the development of a novel T cell expansion
|
This dissertation describes the development of a novel T cell expansion
|
||||||
platform, including the fabrication, quality control, and biological validation
|
platform, including the fabrication, \gls{qc}, and biological validation
|
||||||
of its performance both \invitro{} and \invivo{}. Development of such a system
|
of its performance both \invitro{} and \invivo{}. Development of such a system
|
||||||
would be meaningful even if it only performed as well as current methods, as
|
would be meaningful even if it only performed as well as current methods, as
|
||||||
adding another method to the arsenal of the growing T cell manufacturing
|
adding another method to the arsenal of the growing T cell manufacturing
|
||||||
|
@ -4724,7 +4693,7 @@ function of surface density and the presentation method.
|
||||||
|
|
||||||
\subsection{Reducing Ligand Variance}
|
\subsection{Reducing Ligand Variance}
|
||||||
|
|
||||||
While we have robust quality control steps to quantify each step of the
|
While we have robust \gls{qc} steps to quantify each step of the
|
||||||
\gls{dms} coating process, we still see high variance across time and personnel
|
\gls{dms} coating process, we still see high variance across time and personnel
|
||||||
(\cref{fig:dms_coating}). This is less than ideal for translation.
|
(\cref{fig:dms_coating}). This is less than ideal for translation.
|
||||||
|
|
||||||
|
|
Loading…
Reference in New Issue