From f574b1b331da8093da69a3fca23a98c727385bbe Mon Sep 17 00:00:00 2001 From: ndwarshuis Date: Tue, 3 Aug 2021 13:57:51 -0400 Subject: [PATCH] ADD a bunch of future work fluff --- tex/references.bib | 13 +++++++++++ tex/thesis.tex | 54 +++++++++++++++++++++++++++++++++++++++------- 2 files changed, 59 insertions(+), 8 deletions(-) diff --git a/tex/references.bib b/tex/references.bib index 51f1b11..b02360e 100644 --- a/tex/references.bib +++ b/tex/references.bib @@ -2583,6 +2583,19 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec publisher = {The American Association of Immunologists}, } +@Article{Stephan2014, + author = {Sirkka B Stephan and Alexandria M Taber and Ilona Jileaeva and Ericka P Pegues and Charles L Sentman and Matthias T Stephan}, + journal = {Nature Biotechnology}, + title = {Biopolymer implants enhance the efficacy of adoptive T-cell therapy}, + year = {2014}, + month = {dec}, + number = {1}, + pages = {97--101}, + volume = {33}, + doi = {10.1038/nbt.3104}, + publisher = {Springer Science and Business Media {LLC}}, +} + @Comment{jabref-meta: databaseType:bibtex;} @Comment{jabref-meta: grouping: diff --git a/tex/thesis.tex b/tex/thesis.tex index 21d662d..22d743b 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -163,6 +163,7 @@ \newacronym{qbd}{QbD}{quality-by-design} \newacronym{aws}{AWS}{amazon web services} \newacronym{qpcr}{qPCR}{quantitative polymerase chain reaction} +\newacronym{cstr}{CSTR}{continuously stirred tank bioreactor} %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % SI units for uber nerds @@ -733,7 +734,7 @@ much higher surface area than a traditional flask when matched for volume. Consequently, this means that microcarrier-based cultures can operate with much lower footprints than flask-like systems. Microcarriers also allow cell culture to operate more like a traditional chemical engineering process, wherein a -stirred tank bioreactor may be employed to enhance oxygen transfer, maintain pH, +\gls{cstr} may be employed to enhance oxygen transfer, maintain pH, and continuously supply nutrients\cite{Derakhti2019}. A variety of microcarriers have been designed, primarily differing in their @@ -4315,7 +4316,8 @@ group. \section{future directions} -There are several important next steps to perform with this work: +There are several important next steps to perform with this work, many of which +will be relevent to using this technology in a clinical trial: \subsection{Translation to GMP process} @@ -4341,12 +4343,33 @@ as dynabeads and thus the research-grade proteins used here could be easily replaced. The \gls{snb} is a synthetic small molecule and thus does not have any animal-origin concerns. -\subsection{Mechanistic investigation} +\subsection{mechanistic investigation} -% why do the dms work? -% can we put anything on the dms to enhance their potency? +Despite the improved outcomes in terms of expansion and phenotype relative to +beads, we don't have a good understanding of why they \gls{dms} platform works +as well as it does. Several broad areas remain to be investigated, including the +role of the increased cytokine output (including \il{15} which was explored to +some extent in this work), the role of cells on the interior of the \gls{dms} +relative to those outside the \gls{dms}, and the role of the physical surface +properties of the \gls{dms} (including the morphology and the stiffness). -\subsection{Assessing performance using unhealthy donors} +\subsection{additional ligands and signals on the DMSs} + +In this work we only explored the use of \acd{3} and \acd{28} \glspl{mab} coated +on the surface of the \gls{dms}. The chemistry used for the \gls{dms} is very +general, and any molecule or protein that could be engineered with a biotin +ligand could be attached without any further modification. There are many other +ligands that could have profound effects on the expansion and quality of T cells +which may be utilized. The simplest next step is to simply vary the ratio of +\acd{3} and \acd{28} signal. Another obvious example is to attach +\il{15}/\il{15R$\upalpha$} complexes to the surface to mimic \textit{trans} +presentation from other cell types\cite{Stonier2010}. Other adhesion ligands or +peptides such as GFOGER could be used to stimulate T cells and provide more +motility on the \glspl{dms}\cite{Stephan2014}. Finally, viral delivery systems +could theoretically be attached to the \gls{dms}, greatly simplifying the +transduction step. + +\subsection{assessing performance using unhealthy donors} All the work presented in this dissertation was performed using healthy donors. This was mostly due to the fact that it was much easier to obtain healthy donor @@ -4360,8 +4383,23 @@ expansion technology given that even in healthy donors, we observed the \subsection{translation to bioreactors} -% use dms in non-static bioreactors such as wave by first activating in a static -% environment +In this work we performed some preliminary experiments demonstrating that the +\gls{dms} platform can work in a Grex bioreactor. While an important first step, +more work needs to be done to optimize how this system will or can work in a +scalable environment using bioreactors. There are several paths to explore. +Firstly, the Grex itself has additional automation accessories which could be +tested, which would allow continuous media exchange and cytokine +administration. While this is an improvement from the work done here, it is +still a Grex and has all the disadvantages of an open system. Secondly, other +static bioreactors such as the Quantum hollow fiber bioreactor (Terumo) could be +explored. Essentially the \gls{dms} would be an additional matrix that could be +supplied to this system which would enhance its compatibility with T cells. +Finally, suspension bioreactors such as the classic \gls{cstr} or WAVE +bioreactors could be tried. The caveat with these is that the T cells only seem +to be loosely attached to the \gls{dms} throughout culture, so an initial +activation/transduction step in static culture might be necessary before moving +to a suspension system (alternatively the \gls{dms} could be coated with +additional adhesion ligands to make the T cells attach more strongly). \onecolumn \clearpage