From 8f6319c22309624f6818ed73a1672de0ebae1973 Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Thu, 22 Jul 2021 13:23:44 -0400 Subject: [PATCH] ADD hypotheis --- tex/thesis.tex | 40 +++++++++++++++++++++++++++++----------- 1 file changed, 29 insertions(+), 11 deletions(-) diff --git a/tex/thesis.tex b/tex/thesis.tex index 6244ffc..113bea9 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -55,6 +55,9 @@ \newacronym{dc}{DC}{dendritic cell} \newacronym{il2}{IL2}{interleukin 2} +%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% +% my commands + \newcommand{\mytitle}{ \Large{ \textbf{ @@ -73,6 +76,15 @@ \end{flushleft} } +\newcommand{\invivo}{\textit{in vivo}} + +\newcommand{\invitro}{\textit{in vitro}} + +\newcommand{\exvivo}{\textit{ex vivo}} + +%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% +% my environments + \newenvironment{mytitlepage}{ \begin{singlespace} \begin{center} @@ -82,6 +94,9 @@ \end{singlespace} } +%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% +% document + \begin{document} \begin{titlepage} @@ -191,7 +206,7 @@ Thank you to Lex Fridman and Devin Townsend for being awesome and inspirational. manufacturing large numbers of high quality cells remains challenging. Currently approved T cell expansion technologies involve anti-CD3 and CD28 \glspl{mab}, usually mounted on magnetic beads. This method fails to recapitulate many key -signals found \textit{in vivo} and is also heavily licensed by a few companies, +signals found \invivo{} and is also heavily licensed by a few companies, limiting its long-term usefulness to manufactures and clinicians. Furthermore, we understand that highly potent T cells are generally less-differentiated subtypes such as central memory and stem memory T cells. Despite this @@ -208,7 +223,7 @@ CD4+ T cells, and showing compatibility with existing \gls{car} transduction methods. In aim 2, we use \gls{doe} methodology to optimize the \gls{dms} platform, and develop a computational pipeline to identify and model the effect of measurable \glspl{cqa} and \glspl{cpp} on the final product. In aim 3, we -demonstrate the effectiveness of the \gls{dms} platform \textit{in vivo}. This +demonstrate the effectiveness of the \gls{dms} platform \invivo{}. This thesis lays the groundwork for a novel T cell expansion method which can be used in a clinical setting, and also provides a path toward optimizing for product quality in an industrial setting. @@ -240,6 +255,8 @@ quality in an industrial setting. \chapter{introduction} +\section*{overview} + T cell-based immunotherapies have received great interest from clinicians and industry due to their potential to treat, and often cure, cancer and other diseases\cite{Fesnak2016,Rosenberg2015}. In 2017, Novartis and Kite Pharma @@ -253,14 +270,14 @@ superparamagnetic, iron-based microbeads (Invitrogen Dynabead, Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers (Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017, Bashour2015}. These strategies overlook many of the signaling components present in the -secondary lymphoid organs where T cells expand in vivo. Typically, T cells are +secondary lymphoid organs where T cells expand \invivo{}. Typically, T cells are activated under close cell-cell contact, which allows for efficient autocrine/paracrine signaling via growth-stimulating cytokines such as \gls{il2}. Additionally, the lymphoid tissues are comprised of \gls{ecm} components such as collagen, which provide signals to upregulate proliferation, cytokine production, and pro-survival pathways\cite{Gendron2003, Ohtani2008, Boisvert2007, Ben-Horin2004}. We hypothesized that culture conditions that -better mimic these in vivo expansion conditions of T cells, can significantly +better mimic these \invivo{} expansion conditions of T cells, can significantly improve the quality and quantity of manufactured T cells and provide better control on the resulting T cell phenotype. @@ -278,7 +295,7 @@ Matrigel\cite{Rio2018} or 3d-printed lattices\cite{Delalat2017}, ellipsoid beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms} beads\cite{Lambert2017} that respectively recapitulate the cellular membrane, large interfacial contact area, 3D-structure, or soft surfaces T cells normally -experience in vivo. While these have been shown to provide superior expansion +experience \invivo{}. While these have been shown to provide superior expansion compared to traditional microbeads, none of these methods has been able to show preferential expansion of functional naïve/memory and CD4 T cell populations. Generally, T cells with a lower differentiation state such as naïve and memory @@ -316,18 +333,19 @@ only provide superior expansion, but consistently provide a higher frequency of naïve/memory and CD4 T cells (CCR7+CD62L+) across multiple donors. We also demonstrate functional cytotoxicity using a CD19 \gls{car} and a superior performance, even at a lower \gls{car} T cell dose, of \gls{dms}-expanded -\gls{car}-T cells in vivo in a mouse xenograft model of human B cell \gls{all}. +\gls{car}-T cells \invivo{} in a mouse xenograft model of human B cell \gls{all}. Our results indicate that \glspl{dms} provide a robust and scalable platform for manufacturing therapeutic T cells with higher naïve/memory phenotype and more balanced CD4+ T cell content. -\section*{overview} - -Insert overview here - \section*{hypothesis} -Insert hypothesis here +The hypothesis of this dissertation was that using \glspl{dms} created from +off-the-shelf microcarriers and coated with activating \glspl{mab} would lead to +higher quantity and quality T cells as compared to state-of-the-art bead-based +expansion. The objective of this dissertation was to develop this platform, test +its effectiveness both \invivo{} and \invivo{}, and develop computational +pipelines that could be used in a manufacturing environment. \section*{specific aims} \subsection*{aim 1}